A multivesicular liposome (MVL) is a liposomal vehicle designed to achieve sustained release characteristics for drugs with short half-lives. For example, a commercial MVL formulation of bupivacaine has been approved by the U.S. Food and Drug Administration for local and regional analgesia. For complex formulations like those containing MVLs, challenges in developing an in vitro release testing (IVRT) method may hinder generic development and regulatory approval. In this study, we developed an accelerated rotator-based IVRT method with the ability to discriminate bupivacaine MVLs with different quality attributes. Three IVRT experimental setups including mesh tube, horizontal shaker, and vertical rotator were screened to ensure that at least 50% of bupivacaine can release from MVLs in 24h. Sample dilution factors, incubation temperature, and the release media pH were optimized for the IVRT. The reproducibility of the developed IVRT method was validated with commercial bupivacaine MVLs. The discriminative capacity was assessed via comparing commercial and compromised bupivacaine MVL formulations. The rotator-based release setup was chosen due to the capability to obtain 70% of drug release within 24h. The optimized testing conditions were chosen with a 50-fold dilution factor, a temperature of 37ºC, and a media pH of 7.4. An accelerated rotator-based IVRT method for bupivacaine MVLs was developed in this study, with the discriminatory ability to distinguish between formulations of different qualities. The developed IVRT method was a robust tool for generic development of MVL based formulations.