Benzodiazepines represent a large and important class of psychotherapeutic agents acting on the central nervous system. Due to their tranquilizer, anti-depressive and sedative properties they have become the drugs of choice for the treatment of anxiety, sleep disorders, status epilepsy, insomnia, frequent nocturnal, early morning awakenings or other convulsive disorders. This book compromises brief discussion on structure, properties and the effects of benzodiazepines, comprehensive review on sample preparation and determination of these drugs in biological samples by HPLC methods to be helpful for the researchers. Two new validated HPLC methods for the determination of 1,4-benzodiazepines and an investigation on stability of six commonly prescribed 1,4-benzodiazepines in biological samples are included. This book may be advantageous for routine determination of these drugs in forensic and clinical investigations, and applicable for the quantitative estimation in quality control in pharmaceutical formulations.