In urine drug testing for pain management, dilute and shoot is a common specimen preparation method. Urine contains the parent form of drugs, as well as metabolites. Beta-glucuronidases are enzymes that are used to cleave glucuronide conjugates off the drug metabolite, converting it back into the parent drug, allowing detection of the free drug. There are several different sources of beta-glucuronidase enzymes on the market today, including abalone, red abalone, limpet, and recombinant organisms. This research evaluates beta-glucuronidase enzymes from different companies and sources to determine the enzyme that has the best hydrolysis efficiency. Hydrolysis efficiency is calculated based on the actual conversion of conjugated metabolite to parent drug when specimens are fortified with a known concentration of conjugated metabolite. Six different enzyme sources were used, along with six different drug-glucuronide conjugates, including the drug classes of opioids, benzodiazepines, and cannabinoids. Guidelines were established to ascertain the most efficient and affordable enzyme. The guidelines stated that there could not be less than 40% conversion of the glucuronide conjugates to the target drug, it must work well across multiple drug classes and be cost efficient for a laboratory. Once the results were analyzed using these guidelines, are combinant enzyme was considered to be the top enzyme of the six sources that were used in this research.