The diphenol curcumin from turmeric is rapidly metabolized into phase II conjugates following oral administration, resulting in negligible plasma concentration of the free compound which is considered the bioactive form. Total plasma concentration of curcumin is often quantified after treatment with β-glucuronidase to hydrolyze curcumin-glucuronide, the most abundant conjugate in vivo. The efficiency of enzymatic hydrolysis has not been tested.Using LC-MS analyses we compared the efficiency of β-glucuronidase and sulfatase from Helix pomatia to hydrolyze curcumin conjugates in human and mouse plasma after oral administration of turmeric. Both β-glucuronidase and sulfatase completely hydrolyzed curcumin-glucuronide. Unexpectedly, β-glucuronidase hydrolysis was incomplete, affording a large amount of curcumin-sulfate whereas sulfatase hydrolyzed both glucuronide and sulfate conjugates. With sulfatase, the concentration of free curcumin was doubled in human and increased in mouse plasma compared to β-glucuronidase treatment. Incomplete hydrolysis by β-glucuronidase suggested the presence of mixed glucuronide-sulfate conjugates. LC-MS-based searches detected diglucuronide, disulfate and mixed sulfate-glucuronide and sulfate-diglucuronide conjugates in plasma that likely contribute to the increase of free curcumin upon sulfatase treatment.β-Glucuronidase incompletely hydrolyzes complex sulfate-containing conjugates that appear to be major metabolites, resulting in an underestimation of the total plasma concentration of curcumin. This article is protected by copyright. All rights reserved.