A rapid, simple and specific gas chromatographic method has been developed to measure the differentiating agent hexamethylene bisacetamide (HMBA) in biological samples. Addition of a homolog as an internal standard, ultrafiltration and then direct packed column GC-FID analysis of the ultrafiltrate gives a detection limit of less than 50 μg/ml (0.25 mM) for HMBA in plasma or urine. Ultrafiltration is quantitative and assay precision is better than 4.3% for the 1–5 mM range. This method has been applied to determine the bolus dose pharmacokinetics and disposition of HMBA in a single small animal such as a rat. The developed assay should be suitable for therapeutic monitoring of human patients undergoing HMBA treatment.