Background and Objective: Optical reflectance spectroscopy is a non-invasive technique for optical characterization of biological samples. Any alteration in a cell from normal or carcinogenic causes will change its refractive index. The aim of this study is to develop a computerized program for extraction of a refractive index of normal and cancerous skin cell lines, including melanoma, fibroblast, and adipose cells, using visible near-infrared reflectance spectra and the Kramers-Kronig (K-K) relations. Materials and Method: A fiber optic reflectance spectrometer in visible near-infrared wavelength was used for spectrum acquisition in an in vitro study. Human skin cell lines for melanoma (A375), fibroblast, and adipose sample were cultured for optical spectroscopy. Following data acquisition, an analytical MATLAB code was developed to run the K-K relations. The program was validated for three biological samples using an Abbe refractometer. Results: The validation error (below 5%) and determination of changes in the refractive index of melanoma, normal fibroblasts, and adipose skin cells was carried out at wavelengths of 450-950 nm. The refractive index of melanoma was 1.59270 ± 0.0550 at 450 nm, the minimum amount of 1.27790 ± 0.0550 to 1.321 ± 0.0550 at 620 nm, and rose sharply to 1.44321 ± 0.0550 at 935 nm. The respective results for fibroblast and adipose tissue cells were 1.33282 ± 0.0134 and 1.28345 ± 0.0163 at 450 nm with an increasing trend to 1.30494 ± 0.0135 and 1.26716 ± 0.0163 at 935 nm. Conclusion: Refractive index characteristics show potential for cancer screening and diagnosis. The results show that optical spectroscopy is a promising, non-invasive tool for assessment of the refractive index of living biological cells in in vitro settings. Tracking changes in the refractive index allows screening of normal and abnormal cells for probable alterations in a non-invasive label-free method. Lasers Surg. Med.