Busulfan is widely used in bone marrow transplantation. Increased area under the plasma concentration curve has been shown to correlate with venoocclusive disease, which occurs in approximately 20% of these patients. The authors developed a high performance liquid chromatography assay for the determination of plasma busulfan concentration using ultraviolet detection and a single-step derivatization and extraction. The absolute retention times of busulfan and the internal standard were 2.8 and 5.6 min, respectively. The assay possessed linearity up to 200 mumol/l, sensitivity to at least 0.2 mumol/l, average recovery of 101%, and run-to-run precision (n = 34) of < 7%. Furthermore, the assay proved to be free of interference from 59 medications and correlated highly with a validated method employing gas chromatography with electron capture detection (slope = 0.90, intercept = 0.17, r = 0.98, n = 31). The authors conclude that the method described here is ideally suited for the therapeutic monitoring of busulfan.