Abstract An automated gradient high-performance liquid chromatographic method using a column-switching technique was developed in order to determine and quantify midazolam (separated from the metabolite α-hydroximidazolam) in human plasma. After dilution with an internal standard (flurazepam) solution, containing 20% acetonitrile, 400 μl of the plasma samples were injected onto a precolumn (17 × 4.6 mm I.D., C 18 Corasil 37–53 μm) and retained. Proteins and polar plasma components were washed out using a 0.1 M sodium hydroxide solution, followed by an equilibration with a phosphate buffer of pH 8.0. After column-switching midazolam and flurazepam were eluted and transferred to the analytical column (RP-select B) in the backflush mode, separated by gradient elution and detected at 230 nm by ultraviolet detection. Precision of replicate analyses on the same day was 1.5% for midazolam and 0.7% for flurazepam. Recovery of midazolam was in the range 80–89% and the detection limit was 2 ng/ml plasma.