Attaining capability of label-free optical characterization of tissues will offer methodological advancement and possibilities for early clinical detection, which is of paramount importance in treating patients under clinical setups, for example, cancer. Here, we demonstrate the potential of autofluorescence exhibited by tissues as an enabling microscopic strategy to achieve high-resolution imagery data offering a wealth of clinically relevant information including possibility of three-dimensional rendering. Furthermore, we elucidate the use of analytic tools to extract numerical read-outs from such data with further implications in histopathology, pharmaceutics, toxicology, and screening purposes. This study summarizes the results obtained through a systematic autofluorescence-based investigation on murine and porcine gut tissues with an example of applying the technique in nanotoxicology. The study provides with a methodological roadmap toward developing a fast, effective, and robust platform enabling in-depth optical characterization of tissues.