Rapid developments in the field of organotypic cultures have generated a growing need for effective and non-invasive methods for quality control during tissue development. In this study, we correlate metabolic changes with epidermal differentiation and demonstrate that multiphoton microscopy with fluorescence lifetime imaging (MPM-FLIM) can be applied to monitor epidermal differentiation of keratinocytes with respect to proliferative and differentiated states. In a 2D keratinocyte tissue culture model, increased expression of differentiation markers keratin-1 and keratin-10 was induced with calcium supplementation. An accompanying shift from glycolysis to mitochondrial respiration was detected in metabolic flux assays. Analysis of MPM-FLIM images acquired at 750 nm and 900 nm excitation revealed a decreased relative fraction of intracellular NADH and FAD after high calcium treatment, consistent with increased oxidative phosphorylation. Epidermal differentiation could be monitored over a 96 h period. Discrimination analysis based on k-means clustering generated clusters that correlated well with the duration of high Ca