Chronic wounds are difficult to diagnose and characterize due to a lack of quantitative biomarkers. Label-free multiphoton microscopy has emerged as a useful imaging modality capable of quantifying changes in cellular metabolism using an optical redox ratio of FAD/(NADH+FAD) autofluorescence. However, the utility of an optical redox ratio for long-term in vivo monitoring of tissue metabolism has not been robustly evaluated. In this study, we demonstrate how multiphoton microscopy can be used to monitor changes in the metabolism of individual full-thickness skin wounds in vivo. 3D optical redox ratio maps and NADH fluorescence lifetime images identify differences between diabetic and control mice during the re-epithelialization of wounds. These metabolic changes are associated with a transient increase in keratinocyte proliferation at the wound edge. Our study demonstrates that high-resolution, non-invasive autofluorescence imaging can be performed in vivo and that optical redox ratios can serve as quantitative optical biomarkers of impaired wound healing.