A novel in vitro release test methodology for a liposome formulation was developed using a column-switching high-performance liquid chromatography (HPLC) system. Doxorubicin (DXR) liposome formulations were used as a model. A DXR liposome formulation was dispersed into a release medium, and the dispersion fluid was directly injected at predetermined time points into the column-switching HPLC system. To evaluate the release profile, this system can be used for determining the released and encapsulated DXR in the liposome formulation separately. Comparison with a conventional in vitro release test methodology by dialysis revealed that the methodology developed by column-switching HPLC had no rate-limiting process of membrane permeation of the drug (which is occasionally observed in the dialysis method). The in vitro release profiles of DXR liposome formulations were well characterized using the method developed by column-switching HPLC, and different in vitro release characteristics were revealed. The developed method did not require a large amount of sample or a complicated pretreatment. In addition, the developed column-switching HPLC system was applicable for characterization of the encapsulation profile of liposome formulations.