Benzodiazepines (BDZs) belong to a group of substances known for their sedative, antidepressive, muscle relaxant, tranquilizer, hypnotic and anticonvulsant properties. Their determination in biological fluids is essential in clinical assays as well as in forensics and toxicological studies. Researchers focus on the development of rapid, accurate, precise and sensitive methods for the determination of BDZs and their metabolites. A large number of analytical methods using different techniques have been reported, but none can be considered as the method of choice. BDZs are usually present at trace levels (microgram or nanogram per milliliter) in a complex biological matrix and the potentially interfering compounds must be isolated by various extraction techniques before analysis. An extended and comprehensive review is presented herein, focusing on sample preparation (pretreatment and extraction) and HPLC conditions applied by different authors. These methods enable bioanalysts to achieve detection limits down to 1-2 ng/ml using UV/diode array detection, readily available in most laboratories, and better than 1 ng/ml using electron capture detection, which is lower than that obtained using a nitrogen phosphorus detector. MS interfaced with electrospray ionization offered a similar sensitivity, while negative chemical ionization MS or sonic spray ionization MS provided sensitivity down to 0.1 ng/ml.