Although skin is the largest organ of the human body, cutaneous drug metabolism is often overlooked and existing experimental models are insufficiently validated. This proof-of-concept study investigated phase II biotransformation of 11 test substrates in fresh full-thickness human skin explants, a model containing all skin cell types. Results show that skin explants have significant capacity for glucuronidation, sulfation, N-acetylation, catechol methylation, and glutathione conjugation. Novel skin metabolites were identified, including acyl glucuronides of indomethacin and diclofenac, glucuronides of 17β estradiol, N-acetylprocainamide, and methoxy derivatives of 4 nitrocatechol and 2,3 dihydroxynaphthalene. Measured activities for 10 μM substrate incubations spanned a 1000-fold: from the highest 4.758 pmol·mg skin(-1)·h(-1) for p-toluidine N-acetylation to the lowest 0.006 pmol·mg skin(-1)·h(-1) for 17β estradiol 17 glucuronidation. Interindividual variability was 1.4 to 13.0-fold, being the highest for 4 methylumbelliferone and diclofenac glucuronidation. Reaction rates were generally linear up to 4 h, although 24 h incubations enabled detection of metabolites in trace amounts. All reactions were unaffected by the inclusion of co-substrates and freezing of the fresh skin led to loss of glucuronidation activity. The predicted whole skin intrinsic metabolic clearances (CLint,skin) were significantly lower compared to corresponding whole liver intrinsic clearances (CLint,skin) suggesting a relatively limited contribution of the skin to the body's total systemic phase II enzyme-mediated metabolic clearance. Nevertheless, the fresh full-thickness skin explants represent a suitable model to study cutaneous phase II metabolism not only in drug elimination but also in toxicity as formation of acyl glucuronides and sulfate conjugates could play a role in skin adverse reactions.