High-performance liquid chromatography with tandem mass spectrometry has been used for rapid, specific, and sensitive analysis of busulfan in human plasma. Busulfan-d8 was used as internal standard. Analysis was performed on a C18 column (50 mm × 2.1 mm, 3.5-µm particles) with water–methanol 80:20 (v/v) as mobile phase at a flow-rate of 0.30 mL min−1. Detection was by tandem triple–quadrupole mass spectrometry with turbo ion-spray ionization. Linear calibration plots were obtained over the concentration range 1.096–1,096 ng mL−1. The assay is ideally suited to monitoring of busulfan and determination of its pharmacokinetic data.