Amphotericin B (AMB) is still the standard care for systemic fungal infections. This paper describes a sensitive, accurate and simple liquid chromatography-tandem mass spectrometry method to quantify AMB in human or minipig plasma. Samples were prepared through protein precipitation by adding methanol-acetonitrile (1:3, v/v) to either human or minipig plasma. High-performance liquid chromatography separation was conducted on a 10-cm Gemini C18 column with a 7-min gradient of mobile phase comprised of buffer A (0.1% formic acid aqueous solution) and buffer B (methanol-acetonitrile, 2:3, v/v). AMB was detected through multiple reaction monitoring (MRM) with a mass transition of 924.60 → 743.30 and the internal standard paclitaxel was detected through MRM with a mass transition of 854.30 → 286.10. The method had a linear range between 5 and 2500 ng/mL with lower limit of quantitation of 3 ng/mL. The overall recovery was 113 ± 4.06% in human plasma and 94.8 ± 7.38% in minipig plasma. The method has been validated and applied for AMB pharmacokinetic study in both human and minipig plasma.