Benzodiazepines are among the most prescribed drugs for the treatment of a wide spectrum of clinical disorders. They have been used as anticonvulsants, anxiolytics, hypnotics or muscle relaxants with different durations of action. In this paper, the determination of six frequently used benzodiazepines and metabolites: flunitrazepam, oxazepam, alprazolam, chlordiazepoxide, diazepam and desmethyldiazepam in serum was developed by high-performance liquid chromatography (HPLC) with solid phase extraction (SPE). Quantification was performed with gradient elution of a C8 reversedphase column, with an alkaline wateracetonitrile-methanol eluent, free of buffer salts. Carbamazepine was detected as an internal standard by ultraviolet absorbance at 239 nm. The determination of flunitrazepam, oxazepam, alprazolam, chlordiazepoxide, desmethyldiazepam and diazepam was possible in a concentration range of 250-4,000 ng/mL, and the mean recovery by this analysis was 91, 91, 93, 84, 110 and 92%, respectively. This method allows rapid detection, a purity check, identification, and quantitation of the eluting peaks. Sensitivity and specificity data are acceptable. The validated procedure has been applied routinely in forensic toxicological analysis. Chiang Mai Med J 2007;46(1):23-30.