Catheter malfunction in hemodialysis (HD) is increasingly managed with recombinant tissue plasminogen activator (rt-PA, alteplase), though evidence of improved catheter function is lacking. To evaluate the effect of a standardized rt-PA administration protocol on rt-PA usage, catheter function, and adverse events. Observational quality improvement study. Single, urban, community HD unit in Calgary, Alberta. Patients treated with maintenance in-center HD through central venous catheter. Incidence of rt-PA usage, catheter interventions, hospitalizations, and measures of dialysis efficacy. The rt-PA protocol was designed following a consultative and iterative design period with dialysis shareholders, which included focusing on standard objective criteria before use and targeting use to the problematic lumen. Protocol implementation occurred over a 6-month period in 2021. Patient and dialysis data were collected through our regional dialysis electronic health record. Implementation of the rt-PA protocol resulted in decreased rt-PA use (standardized per 100 dialysis sessions) compared to the preprotocol period (incidence rate ratio [IRR] of 0.57, 95% confidence interval [CI]: [0.34, 0.94]). Line procedures were also less frequent (IRR = 0.42, 95% CI: [0.18, 0.89]). Hospitalization rates and measures of dialysis efficacy were similar in both periods. Small sample size with single dialysis center and short duration of follow-up. Implementation of a multidisciplinary designed rt-PA administration protocol decreased incident rt-PA usage.