Approximately half of people with endometriosis experience deep dyspareunia; however, there is no means of objective self-testing of endometriosis-associated deep dyspareunia. The aim of this study was to assess the acceptability, test-retest reliability, and validity of a vaginal insert for a self-assessment of endometriosis-associated deep dyspareunia. Participants were recruited from a tertiary endometriosis center. Inclusion criteria were: 19 to 49years of age, self-reported deep dyspareunia of ≥4 of 10, and surgically confirmed endometriosis. Participants completed 2 self-assessments using the vaginal insert to self-assess tenderness at the right and left pelvic floor, bladder, cervix-uterus, and posterior cul-de-sac (vaginal fornix). The participants recorded tenderness at each pelvic site and completed a questionnaire regarding the acceptability of the vaginal insert to assess deep dyspareunia. Test-retest reliability was assessed by correlating the tenderness scores between the 2 assessment dates. Over a 4-week period, the participants also recorded deep dyspareunia severity at each penetrative vaginal sex encounter. Validity was assessed by correlating vaginal insert tenderness to deep dyspareunia severity, and also to tenderness reported on a prior gynecologic pelvic examination. The main outcome measures were the acceptability index score, tenderness (0-10) at each pelvic site, and prospective deep dyspareunia scores (0-10) over 4 weeks. There were 19 participants (mean age 34±7years) who completed the study. The majority identified as female (94.7%), heterosexual (89.5%), and white (89.5%). The median acceptability index score was 0.72 (interquartile range, 0.66-0.81). For test-retest reliability, the intraclass correlation coefficients were 0.79 (P = .001) for the left pelvic floor, 0.82 (P < .001) for the right pelvic floor, 0.54 (P = .07) for the bladder, 0.89 (P < .001) for the cervix-uterus, and 0.77 (P = .003) for the cul-de-sac. The correlation between the highest self-assessed mean tenderness in each participant and self-reported deep dyspareunia over 4 weeks was r = 0.32, but correlations for each pelvic site varied significantly. Tenderness at each site on prior gynecologist pelvic exam was associated with higher self-assessed mean tenderness with the vaginal insert in each participant (effect sizes = 0.42-0.88). The vaginal insert is acceptable and reliable for the objective self-assessment of endometriosis-associated deep dyspareunia, with initial evidence of validity. A strength was the inclusion of participants who were avoiding sexual activity and a limitation was the small sample size. Future studies with larger sample sizes are required to further establish the validity of the vaginal insert for the self-assessment of endometriosis-associated deep dyspareunia.