This report describes a full-scale experiment on intradermal Ehrlich carcinoma (EC) differentiation in mouse model using NIR spectroscopy in diffuse reflectance mode and chemometric data processing. EC is widely used as an experimental tumor model due to its resemblance with human undifferentiated epithelial tumors and can be applied as a preclinical testing in order to verify the capability of NIR spectroscopy to distinguish cancer from healthy tissues before a clinical research with an aim of creating a new analytical tool for on-line intraoperative tumor margins assessment. The study consists of five steps of NIR spectra measurements: in vivo on the early stage of carcinoma growth; in vivo on the advanced stage of carcinoma growth; in vivo during the surgery; in vitro study of the post-operative materials stored in formalin; in vitro study of the post-operative materials stored in paraffin. It was shown that reliable tumor differentiation with a compact optic fiber probe was possible in all these cases. The classification models were built on two data sets, obtained during in vivo and in vitro measurements; both of them demonstrated 100% specificity and sensitivity.