Interest in the development of optical technologies that have the capability of performing in situ tissue diagnosis without the need for surgical biopsy and processing has been growing. In general, optical diagnostic techniques can be classified into two categories: (1) spectroscopic diagnostics and (2) optical imaging. Spectroscopic diagnostic techniques are used to obtain an entire spectrum of a single tissue site (point-measurement method). On the other hand, optical imaging methods are aimed at recording a two- or three-dimensional image of a sample region. A third category, which combines the two modalities, is currently in an early development phase. This category, referred to as spectral imaging, has been applied to cytomics, fluorescence resonance energy transfer (FRET) analysis, histology, fluorescence microscopy and autofluorescence microscopy. In this study, we combined a multi-photon microscope with a sensitive prism-based spectrograph and employed it for intrinsic emission spectral imaging microscopy of in vivo mouse skin tissues. We show results on: (1) spectral image RGB real-color visualization; (2) tissue layer discrimination using spectral signatures; (3) depth-resolved skin tissue spectral imaging; and (4) tissue component determination by spectral (linear) unmixing.