Skin has a highly regulated pH environment of around pH 7.2 but with an acid barrier mantle of around pH 5.5. Trauma, inflammation, and infection are all thought to disrupt this pH environment but the lack of a non-invasive technique to measure pH within discrete locations within skin has hindered investigating what role pH plays in wound healing. In this study, a confocal Raman microspectroscopy method was used for measuring pH in a 3D tissue engineered model of human skin (TE-skin) and evaluated for its ability to detect changes in pH in response to wounding, inflammation and bacterial infection. The state of protonation of phosphate groups within the TE-skin was used to indicate pH in a non-destructive manner exploring depths of skin from the stratum corneum to 600 microns into the dermis. Deliberate wounding or inflammation (induced by IL-17) resulted in a loss of the acid mantle. Detailed scanning of TE-skin infected with Staphylococcus aureus or Pseudomonas aeruginosa revealed heterogeneous pH microenvironments ranging in size from 10 × 10 to 50 × 100 microns and ranging from pH 5 to 9. These microenvironments were not detected if an average pH for the TE-skin model was used.