Benzodiazepines are sedative/hypnotic drugs routinely prescribed to treat conditions such as anxiety, insomnia, and epilepsy. Patients using these drugs are often monitored to ensure that the drugs are being used properly, as they are common targets of abuse due to their sedative effects. The major metabolic pathway of benzodiazepines includes conjugation with glucuronides. Most laboratories choose to look for the free form of the drug rather than the conjugated metabolite; therefore, analysis of benzodiazepines requires enzyme hydrolysis of glucuronides for accurate detection of the drugs and drug metabolites in urine. A recombinant β-glucuronidase enzyme, IMCSzyme™, was used in this experiment to reduce hydrolysis time. Thomson eXtreme|FV ® 0.2μm PVDF (polyvinylidene fluoride filter vials) were used in place of solid phase extraction to further decrease the preparation time associated with analysis of benzodiazepines. Analysis of liquid chromatography and tandem mass spectrometry (LC/MS/MS) data indicated that 30 minutes of hydrolysis at 55°C was ideal to break the glucuronide linkages associated with benzodiazepine metabolites. In addition, the sample preparation method was changed from a more time consuming Solid Phase Extraction Method (SPE) to a simple filtration method. A three day validation study confirmed that the method may be applied to the analysis of patient samples.