The use of tandem mass spectrometry (MS/MS) for urine drug testing has expanded in recent decades due to an increasing demand for compliance testing and confirmation of positive immunoassay results. While it has been demonstrated to be a robust methodology, MS/MS cannot control all variables associated with sample collection and testing. This review will touch on many factors of targeted MS/MS assays pertaining to drugs, metabolism, and proper method development. Various preanalytical and analytical considerations will be discussed, and best practice guidelines are suggested for controlling sample-to-sample variability.