Amphotericin B (AMB) is a polyene macrolide antibiotic used for treating invasive fungal infections. Liposomal AMB (L-AMB) is a lipid dosage form which reduces the side effects and toxicity of the drug. The quantitation of free AMB (F-AMB) and L-AMB in vivo is important to monitor quality control of the liposomal formulation and to ensure its safety during clinical use. In this study, an original strategy was developed to separately determine F-AMB and L-AMB in rat plasma using LC–MS/MS. F-AMB was analyzed after separation by solid phase extraction, total AMB (T-AMB) was determined after protein precipitation and L-AMB was determined by difference. The method was fully validated. Calibration curves were linear in the ranges 0.7–120μg/mL for T-AMB and 0.2–20μg/mL for F-AMB. Accuracy and precision results were within acceptable variability limits, recoveries were consistent and reproducible, matrix effects were insignificant and analytes were stable under all the storage conditions tested. The method was successfully applied to a pharmacokinetic study in rats administered a single intravenous 6mg/kg dose of L-AMB. The method will allow further clinical studies of L-AMB and provide useful technical support for the assay of other liposomal drug formulations.