LAUSR

&NA; Amphotericin B is an antifungal drug widely used in Intensive Care Units. Therapeutic drug monitoring (TDM) of amphotericin B is recommended for the assessment of toxicity surveillance and treatment optimization. In this paper we described the development and validation of a new Ultra High Performance Liquid Chromatography coupled to Diode Array Detection (UHPLC‐DAD) method for the quantification of Amphotericin B in 200 &mgr;L human plasma over a wide range of concentrations (0.125–10 mg/L). The new method has been validated following international guidelines on bioanalytical method validation and showed high selectivity, high accuracy and precision and high process efficiency. The new UHPLC‐DAD method that we describe is robust, rapid, cost effective and suitable for application to the routine TDM analyses. HighlightsA new method to quantify amphotericin B in human plasma is reported.The new method is based on a rapid protein precipitation and UHPLC‐DAD.The new method is fast and robust with a total runtime of 8 min.The new method allows the accurate and precise quantification of amphotericin B over a wide range of concentrations (0.125–10 mg/L).The new method has been validated on clinical samples derived from pediatric patients with invasive micoses.