A method is described for determining amphotericin B in plasma using second-derivative spectrophotometry after deproteinization. The assay was based on the absorbance at 407.5 nm. The second-derivative spectrum recorded between 350 and 450 nm allowed identification of the analyte and showed absence of drug interference. Only bilirubin interfered at high concentration (> or = 50 mumol l-1. The linear concentration ranges were 0.05 -5.0 mg l-1 (r = 0.999, slope = 2.731, intercept = 0.008). Between-day CV < or = 9.7%, within-day CV < or = 5.5%, analytical recovery close to 100% were suitable for clinical investigations. This method provides better specificity than direct absorbance, is simpler and faster than a high performance liquid chromatography assay and can be used routinely by any laboratory possessing a spectrophotometer with a derivative accessory.