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Potential application of elastic light single-scattering spectroscopy (ELSSS) for differentiating high-grade squamous intraepithelial lesions (HSIL) from non-HSIL tissues was investigated. An ELSSS system was used to acquire spectra from cervix tissues. A single-fiber optical probe with a diameter of 100 μm was used for both delivery and detection of white light to and from the cervix tissue. Spectroscopic measurements were acquired from 95 ex vivo biopsy samples of 60 pap smear positive patients and normal cervix tissue from 10 patients after hysterectomy were used as a negative control group. Spectroscopic results of 95 cervix biopsy were compared to the histopathology of the biopsy samples. Sensitivity and specificity of the ELSSS system in the differentiation of HSIL and non-HSIL tissues are 87.5% and 45.6%, respectively, for the pap smear and colposcopy positive biopsy samples. The ELSSS system has the potential for use in real-time diagnosis of HSIL tissues as an adjunct to Papanicolaou test (pap smear) and colposcopy.