Cervical cancer is the second most common female cancer worldwide. The ability to quantify physiological and morphological changes in the cervix is not only useful in the diagnosis of cervical precancers but also important in aiding the design of cost-effective detection systems for use in developing countries that lack well-established screening and diagnostic programs. We assessed the capability of a diffuse reflectance spectroscopy technique to identify contrasts in optical biomarkers that vary with different grades of cervical intraepithelial neoplasia (CIN) from normal cervical tissues. The technology consists of an optical probe and an instrument (with broadband light source, dispersive element, and detector), and a Monte Carlo algorithm to extract optical biomarker contributions including total hemoglobin (Hb) concentration, Hb saturation, and reduced scattering coefficient from the measured spectra. Among 38 patients and 89 sites examined, 46 squamous normal sites, 18 CIN 1, and 15 CIN 2+ sites were included in the analysis. Total Hb was statistically higher in CIN 2+ (18.3 ± 3.6 µM, mean ± SE) compared with normal (9.58 ± 1.91 µM) and CIN 1 (12.8 ± 2.6 µM), whereas scattering was significantly reduced in CIN 1 (8.3 ± 0.8 cm-1) and CIN 2+ (8.6 ± 1.0 cm-1) compared with normal (10.2 ± 1.1 cm-1). Hemoglobin saturation was not significantly altered in CIN 2+ compared with normal and CIN 1. The difference in total Hb is likely because of stromal angiogenesis, whereas decreased scattering can be attributed to breakdown of collagen network in the cervical stroma.