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Amphotericin B is available in various drug delivery systems such as cholesteryl sulfate complex, as lipid complex, and as liposomal formulation. The separation and measurement of free drug (drug which is not bound with liposomal lipids) and liposomal drug (drug which is entrapped in liposomes) in the human plasma after injection of liposomal Amphotericin B is of prime importance due to toxicity concerns. A robust, specific and sensitive method has been developed to effectively separate and then quantify the free drug and liposomal drug, present in human plasma. This method utilizes solid phase extraction Oasis HLB cartridges, which retains the free drug and the liposomal Amphotericin B was eluted from the cartridge in first step. The eluted liposomal Amphotericin B was then extracted from lipids by protein precipitation method using 2% dimethylsulfoxide (DMSO) in acetonitrile. After separation and extraction, the quantification of free and liposomal fractions of Amphotericin B was performed by HPLC-MS-MS technique. The chromatographic separation was performed using Chromolith Performance RP 18e column. The mobile phase composed of 5 mM ammonium acetate, methanol and acetonitrile and a gradient elution program was used. The calibration curves were found to be linear for free Amphotericin B (0.25-15.0 mu g/ml) and liposomal Amphotericin B (1.0-100.0 mu g/ml). The recovery was about 96% for free Amphotericin B and about 92% for liposomal Amphotericin B. Recoveries were consistent over the linearity ranges defined. The intra-batch and inter-batch accuracy and precision fulfilled the international requirements. The stability of free and liposomal Amphotericin B was assessed Under different storage conditions. (C) 2009 Elsevier B.V. All rights reserved.