© 2020 Elsevier Inc. Several human enzymes of the short-chain dehydrogenase/reductase (SDR) superfamily of proteins exhibit catalytic oxidoreductive activity toward retinoid substrates in vitro. For some retinoid-active enzymes, their physiological significance for retinoid metabolism is supported by phenotypes linked to naturally occurring mutations in human genes or by targeted gene knockout studies of their murine homologs. However, for those enzymes that are not well conserved or display properties different from their murine counterparts, evaluation of their physiological roles can be challenging. Here, we describe the adaptation of stratified organotypic culture of human epidermis for evaluating the contribution of human putative SDR retinol dehydrogenases to biosynthesis of all-trans-retinoic acid in a three-dimensional cellular model highly sensitive to the levels of all-trans-retinol and all-trans-retinoic acid. In addition to providing a valuable readout of metabolic changes occurring in the presence or absence of the enzyme of interest, this model allows for evaluation of the effects of various retinoid and rexinoid therapeutic compounds on retinoic acid signaling, cell growth and differentiation.