A new 96-well disk solid phase extraction sample preparation technique which does not involve vacuum pumps integrated with liquid chromatographic tandem mass spectrometric (LC-MS/MS) was developed for high throughput determination of benzodiazepines (nordiazepam, diazepam, lorazepam and oxazepam). In addition, the method completely allows the re-use of the SPE disk membranes for subsequent analyses after re-conditioning. The method utilizes a robotic autosampler for parallel extractions in a 96-well plate format. Results have been presented for independent extractions from three matrices; phosphate buffer solution, urine, and plasma. Factors affecting data reproducibility, extraction kinetics, sample throughput, and reliability of the system were investigated and optimized. A total time required per sample was 0.94 min using 96-well format. Method reproducibility was < or =9% relative standard deviation for all three matrices. Limits of detection and quantitation recorded were respectively in the range 0.02-0.15 and 0.2-2.0 ng/mL with linearity ranging from 0.2 to 500 ng/mL for all matrices.