A sensitive method was developed for quantitation of the cytotoxic antibiotic l-alanosine in human plasma. Alanosine was extracted from plasma by anion-exchange solid phase extraction, derivatized with dansyl chloride and analyzed by liquid chromatography-tandem mass spectrometry using atmospheric pressure chemical ionization in negative mode. Dansylation led to 50-fold improvement of method sensitivity over non-dansylated alanosine with a resulting 20 ng/ml limit of alanosine quantitation in plasma being achieved. The method was validated and applied for clinical studies of alanosine administered to cancer patients.