Several metabolites are known to be glucuronidated and sulfated in the human body. Monitoring the aglycones with the gluruco-nidated and sulfated metabolites creates six additional masses to monitor on tandem mass spectrometry, which can limit the total number of metabolites being monitored. In addition, the conjugated standards are very expensive and often not available. Herein,we report the use of purifiedβ-glucuronidase (IMCSzyme®) and recombinant arylsulfatase that effectively cleave glucuronidesand sulfates at neutral pH with no detectable CYP activity or esterase activity. The initial studies explored urinary corticosteroidmetabolites on tandem mass spectrometry before and after cleaving glucuronides and sulfates with the purified enzymes. Among corticosteroids, glucuronidated allo-tetrahydrocortisol (aTHF) has been reported to be recalcitrant substrate for enzymatic decon-jugation. We demonstrate rapid hydrolysis within 10 minutes, while the reported 4 hour incubation using crude snail enzyme is in-adequate for recovering the higher levels of a THF glucuronide (1,2).