An improved reversed-phase liquid chromatographic assay for the anticonvulsant clonazepam in human serum is presented. The drug and two internal standards are extracted from serum with n-butyl chloride at pH 9.2, back-extracted into 2 N HCl, and then extracted at pH 11 into a solvent mixture of chloroform/isopropanol (9:1 by volume). The final extract is evaporated to dryness, reconstituted, and injected into a cyanopropylsilane liquid chromatography column with a mobile phase of 0.5 N acetic acid, acetonitrile, and n-butylamine (59:41:0.005 by volume, respectively). Linearity to 400 micrograms/L was demonstrated, and the detection limit was found to be approximately 5 micrograms/L. Relative recovery from serum as compared to water was approximately 100% for clonazepam and the two internal standards, flunitrazepam and desmethyldiazepam. Within-run precision at 40 micrograms/L was demonstrated by a coefficient of variation (CV) of 2.60% (flunitrazepam internal standard) and 2.53% (desmethyldiazepam internal standard). Between-day multiple analyst precision was demonstrated by a CV of 8.98% (mean 10.09 micrograms/L, n = 77) and 6.51% (mean 28.14 micrograms/L, n = 81) using either internal standard. Capacity factor diagrams reveal at least a dual mechanism separation. Chromatograms of the analysis of clonazepam and other benzodiazepines are presented. The extraction has been optimized for selectivity, thereby minimizing interference from drugs and other extraneous compounds. The chromatographic system has been optimized for speed, efficiency, resolution, and stability and avoids the problem of carbamazepine interference that is commonly encountered with other reversed-phase procedures using octylsilane or octadecylsilane columns.