Busulfan is an alkylating agent widely used in combination chemotherapy regimens followed by allogeneic or autologous hematopoietic stem cell transplantation (HSCT). We present a rapid method for assaying busulfan in pharmaceutical preparations using high-performance thin-layer chromatography (HPTLC) and derivatization with 4-nitrobenzylpyridine. The method is accurate and precise and allows quantitation of busulfan in aqueous solutions from 100 to 500 microg/ml. It is suitable for identification. quantitation and stability studies of busulfan in pharmaceutical products, i.e. capsules or infusion bags prepared in a hospital pharmacy.