A relatively rapid and direct method was developed to determine the concentration of amphotericin B in plasma or whole blood. The characteristic maximum emission fluorescence peak observed for amphotericin B at 488nm, after its excitation at 358nm, was used as the basis for its quantitation. Amphotericin B was extracted and plasma or whole blood proteins were precipitated initially with 50% methanol. Residual plasma proteins were precipitated with 5% trichloroacetic acid to reduce their fluorimetric interference to a minimum. Concentrations in the range 0.1 to 4.0μM could be quantitated relatively rapidly by this method. The method was applied successfully to the determination of amphotericin B in its pharmaceutical dosage form in spiked human plasma or whole blood and in the plasma of a patient treated with amphotericin B.