The objective of this study was to assess the diagnostic potential of synchronous fluorescence (SF) spectroscopy (SFS) technique for the detection and characterization of normal and different malignancy stages of moderately differentiated squamous cell carcinoma (MDSCC), poorly differentiated squamous cell carcinoma (PDSCC) cervical tissues. SF spectra were measured from 45 biopsies from 30 patients in vitro. Characteristic, highly resolved peaks and significant spectral differences between normal and MDSCC, PDSCC cervical tissues were obtained. Nine potential ratios were calculated and used as input variables for a discriminant analysis across different groups. The potentiality of the SFS technique was estimated by two discriminant analyses. Discriminant analysis I performed across normal and abnormal (including MDSCC and PDSCC) cervical tissues classified as 100% both original and the cross-validated grouped cases. In discriminant analysis II performed across the three groups, normal, MDSCC and PDSCC, 100% of both original and the cross-validated grouped cases were correctly classified. Using the SFS technique, one can obtain all the key biochemical markers such as tryptophan, collagen, hemoglobin, reduced form of nicotinamide adenine dinucleotide and flavin adenine dinucleotide in a single scan and hence they can be targeted as tumor markers in the detection of normal from abnormal cervical tissues.