Pain management laboratories analyze biological fluids (urine, saliva or blood) from patients treated for chronic pain to ensure compliance and to detect undisclosed drug use. The quantitation of multi-panel drugs in urine and tissues utilizes β-glucuronidase to cleave the glucuronic acid and liberate the parent drug for mass spectrometry analysis. This work focuses on the comparison of three different, purified and commercially available β-glucuronidases across 83 patient urine samples. One enzyme is genetically modified, expressed in bacteria and the other two enzymes are purified from abalone. The results indicate that the source of β-glucuronidase plays an important role in substrate specificity which in turn dictates hydrolysis efficiency. Contaminants in the enzyme solutions also interfere with analyte detection. Altogether, these factors impact precision and accuracy of data interpretation, leading up to 13% positive/negative disagreement.