It is widely accepted in urine drug testing (UDT) that enzymatic hydrolysis is the preferred practice to increase target analyte detection limits, minimize cost and improve clinical utility of the assay. The addition of beta-glucuronidase for hydrolysis of urine samples has been vastly adapted in labs across the globe. In today’s UDT labs, cutting lab expenses continues to be a focal point. To that end, “dilute and shoot” sample analysis has become more common. Understanding the significance of this impact on LC/MS robustness, labs must balance the sample dilution factor, analyte sensitivity, and avoid injecting added proteins from the beta-glucuronidase into the system. Large dilutions or protein precipitation with centrifugation are standard protocols in clinical and forensic laboratories alike. Achieving the necessary cutoff levels robustly becomes a challenge, especially without the luxury of high-end LC-MS/MS systems.