For the determination of amphotericin B in serum a high-performance liquid chromatography (HPLC) method using simple protein precipitation and an isocratic mobile phase as well as a plate diffusion bioassay are described. Using 5-fluorocytosine-resistant strain of Candida albicans and peptone in medium as an antagonist of 5-fluorocytosine allows simple measurement of amphotericin B both in the absence and in the presence of 5-fluorocytosine. The correlation coefficient (r) between bioassay and HPLC runs is 0.88. Generally, the biological determination gives slightly higher values of amphotericin B than the HPLC method. Both methods are useful for amphotericin B drug monitoring.