Histological analysis, which is used to detect and diagnose most tissue alterations, requires an invasive biopsy procedure and a time-consuming tissue treatment, which limit its efficiency in providing rapid, cost-effective diagnosis and hinder the longitudinal study of tissue alteration. To address these limitations, we have developed a novel procedure, using the features of elastic-scattering spectroscopy, for a real-time, non-invasive analysis of tissues. We have tested whether this approach can detect in vivo changes in mouse skin induced by a single exposure to either complete Freund's adjuvant or 12-O-tetradecanoylphorbol-13-acetate, two drugs known to induce discrete alterations of epidermis and dermis, without obvious changes on the skin surface. Here we report that the evaluation of localized absorption and reduced scattering coefficients permitted the detection of changes in skin regions that showed histological alterations, but not in regions which failed to be modified by the drugs. Results show that the optical in vivo analysis of small regions has sufficient specificity and sensitivity to detect minimal alterations of superficial tissues. In view of the prominent involvement of mucosal alterations in most human diseases, including carcinomas, the method provides a useful complement to standard biopsy, notably for the in vivo screening of early in situ epithelial alterations.