Background A single-institution longitudinal radiation oncology curriculum for medical assistants was shown to be sustainable over multiple years and increased job satisfaction.1 We aimed to assess the sustainability of a multi-institutional expansion of a monthly web-based longitudinal curriculum, and its impact on attendees' confidence and knowledge. Methods Four institutions were recruited, and non-physician staff participants were asked to participate. Between September 2022 and December 2022, participants completed 3 of 15 planned sessions including (1) radiation oncology clinical workflows, (2) medications and vital signs, and (3) prostate cancer. Before and after each session, participants completed surveys about their confidence in knowledge of session topics on a likert-5 point scale (1="very unconfident" to 5= "very confident") and completed knowledge tests. Results of confidence surveys were dichotomized (≥4 vs. ≤3). Surveys and tests were summarized using descriptive statistics and paired t-test. Results Twenty-four of thirty-five (69%) participants report clinical roles (4-7 per institution). Ten (42%) were medical assistants (MA), 8 (33%) were allied health professionals including registered nurses (RN), licensed vocational nurses (LVNs), or advanced practice providers (APP). In pre-session surveys, half of respondents were confident (Likert ≥4) in their understanding of consult notes or knowledge of common medications or abnormal vital signs. Less than half were confident in their ability to understand or use "one-liners" (∼32%) or knowledge of common systemic therapies (29%), medication side effects (41%), risk factors for prostate cancer (25%), or how prostate cancer is staged or managed (38%). On average, respondents were more likely to report confidence in their knowledge after session one (45% to 73%, p<0.01), two (52% to 83%, p<0.01), and three (40% to 95%, p<0.01). On pre-/post-knowledge tests, participants scored similarly for session one (84% vs. 79%, p=0.17) but tended to have increased scores after session two (62% to 74%, p=0.19) and three (38% to 60%, p=0.09), though these differences were not statistically significant. Most participants (85%) considered the amount and level of content appropriate despite the mixed clinical roles of participants. Discussion We report the feasibility of expanding a longitudinal interprofessional curriculum to four institutions, the successful integration of medical assistants, nurses, and advanced practice providers, and incorporation of objective knowledge tests. Participants reported increased confidence in their knowledge after each session and tended to score higher on post-assessments. Future directions include additional monthly disease-site based sessions, incorporation of online asynchronous learning, and incorporation of spaced-repetition of content into assessment to promote knowledge retention. A single-institution longitudinal radiation oncology curriculum for medical assistants was shown to be sustainable over multiple years and increased job satisfaction.1 We aimed to assess the sustainability of a multi-institutional expansion of a monthly web-based longitudinal curriculum, and its impact on attendees' confidence and knowledge. Four institutions were recruited, and non-physician staff participants were asked to participate. Between September 2022 and December 2022, participants completed 3 of 15 planned sessions including (1) radiation oncology clinical workflows, (2) medications and vital signs, and (3) prostate cancer. Before and after each session, participants completed surveys about their confidence in knowledge of session topics on a likert-5 point scale (1="very unconfident" to 5= "very confident") and completed knowledge tests. Results of confidence surveys were dichotomized (≥4 vs. ≤3). Surveys and tests were summarized using descriptive statistics and paired t-test. Twenty-four of thirty-five (69%) participants report clinical roles (4-7 per institution). Ten (42%) were medical assistants (MA), 8 (33%) were allied health professionals including registered nurses (RN), licensed vocational nurses (LVNs), or advanced practice providers (APP). In pre-session surveys, half of respondents were confident (Likert ≥4) in their understanding of consult notes or knowledge of common medications or abnormal vital signs. Less than half were confident in their ability to understand or use "one-liners" (∼32%) or knowledge of common systemic therapies (29%), medication side effects (41%), risk factors for prostate cancer (25%), or how prostate cancer is staged or managed (38%). On average, respondents were more likely to report confidence in their knowledge after session one (45% to 73%, p<0.01), two (52% to 83%, p<0.01), and three (40% to 95%, p<0.01). On pre-/post-knowledge tests, participants scored similarly for session one (84% vs. 79%, p=0.17) but tended to have increased scores after session two (62% to 74%, p=0.19) and three (38% to 60%, p=0.09), though these differences were not statistically significant. Most participants (85%) considered the amount and level of content appropriate despite the mixed clinical roles of participants. We report the feasibility of expanding a longitudinal interprofessional curriculum to four institutions, the successful integration of medical assistants, nurses, and advanced practice providers, and incorporation of objective knowledge tests. Participants reported increased confidence in their knowledge after each session and tended to score higher on post-assessments. Future directions include additional monthly disease-site based sessions, incorporation of online asynchronous learning, and incorporation of spaced-repetition of content into assessment to promote knowledge retention.