Biological samples are analyzed for benzodiazepines in a single isocratic analysis using a chromatographic column system containing an immobilized enzyme reactor which cleaves glucuronic acid-conjugated benzodiazepines, an anion exchange column, a hydrophobic cation exchange column and a reverse-phase analytical column. Preferred methods of performing the analysis further involve the use of a hydrophobic cation exchange precolumn prior to the anion exchange column. The system readily lends itself to automation, automatic periodic sampling and benzodiazepine identification and quantification. The system is particularly well adapted to the determination and identification of benzodiazepines in urine samples.