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Purpose: Rituximab is prone to infusion-related reactions, which commonly requires desensitization to maintain its administration. Conventional desensitization protocols are using multistep infusion by diluting solutions. However, the process of diluting drugs and stepwise delivery needs additional time and effort. The objective of this study was to investigate the safety and efficacy of a nondiluting, one-bag protocol of rituximab desensitization. Methods: A retrospective study was performed by reviewing the medical records of patients who underwent rituximab desensitization between 2009 and 2018. The completion rate, occurrence and severity of breakthrough reactions (BTR), and time required to complete the therapy were compared between one-bag protocol and multibag protocol. Results were analyzed by generalized estimation equation method, and odds ratios (ORs) of completion rate and BTR incidence were estimated. Results: Total 190 cases of desensitization therapy were performed in 49 patients; the incidence of BTR was 16.84% and the overall completion rate was 96.32%. No significant difference in completion rate was found (OR, 3.58; 95% confidence interval [CI], 0.79–16.38) and there was no significant difference in BTR incidence (OR, 0.81; 95% CI, 0.23–2.82) in one-bag protocol. BTR in the one-bag protocol tended to occur even through entire steps, whereas most of the BTR in the multibag protocol occurred at later steps of the process. The average time spent in the desensitization was 60 minutes shorter in the one-bag than the multibag protocol (258.15 minutes vs. 329.81 minutes, P<0.001). Conclusion: One-bag desensitization protocol showed no significant difference in safety and efficiency compared to the conventional multibag protocol, with shortening the time required for completion.