Busulfan is a chemotherapy drug widely used as part of conditioning regimens for patients undergoing bone marrow transplantation (BMT). Challenges of busulfan treatment include a narrow therapeutic window and wide inter- and intra-patient variability. Inappropriately low drug levels lead to relapse and even graft rejection, while higher doses frequently have toxic and sometimes fatal consequences. Maintenance of plasma busulfan concentrations using repeated measurements and proper adjustment of dosage can reduce busulfan-related toxicity and improve treatment outcomes. We describe a rapid (2-minute total analysis time per sample) and simple method for accurate and precise busulfan concentration determination in plasma samples (100 �L) using high performance liquid chromatography combined with electrospray positive ionization tandem mass spectrometry (HPLC-ESI-MS/MS). Busulfan is isolated from plasma after internal standard (busulfan-D8 )–methanol extraction, dilution with mobile phase (ammonium acetate–formic acid–water), and centrifugation. The supernatant plasma is injected onto the HPLC-ESI-MS/MS and quantified using a six-point standard curve. The assay is linear from 0.025 �g/mL (~0.1 �mol/L) to at least 6.2 �g/mL (~25 �mol/L) with precisions of <5% over the entire range.