This technical specification (TS) specifies the methodology to measure total, encapsulated, and free drug concentrations in poly-ethylene glycol functionalized (PEGylated) doxorubicin hydrochloride liposomal formulations. Total drug is quantitated by reversed phase high performance liquid chromatography (RP-HPLC) with ultra-violet (UV) extinction or fluorescence (FL) intensity detection. Suitable separation techniques such as ultrafiltration, solid phase extraction, or asymmetric-flow field-flow fractionation (AF4) can be employed to separate free drug from encapsulated drug. Suitable techniques (e.g., RP-HPLC, UV or FL spectroscopy) can be used to measure free drug concentration. The methodology presented in this technical specification (TS) utilizes RP-HPLC with ultra-violet detection to assess total doxorubicin concentration in the sample. An ultrafiltration process is subsequently used to separate free doxorubicin from encapsulated doxorubicin and the free doxorubicin concentration is measured by fluorescence spectroscopy. Sample preparation and representative RP-HPLC conditions are given based on a commercially available PEGylated doxorubicin hydrochloride liposome formulation which contains total doxorubicin hydrochloride at a concentration of 2 mg/mL with >90% encapsulated within the liposome. The methodology of this TS may be adaptable and applicable to other liposomal formulations with similar physicochemical properties, including morphology, size, charge, or drug properties; however, performance has not been evaluated for these applications.