Colorectal cancer is the second most frequently diagnosed cancer worldwide. Conventional diagnostics methods of colorectal cancer, can detect it in advanced stage. Spectroscopic methods, including Raman spectroscopy and imaging, are becoming more and more popular in medical applications, and allow fast, precise and unambiguous differentiation of healthy and cancerous samples. the most important advantage of Raman spectroscopy is ability to identify biomarkers that help in differentiation of healthy and cancerous cells based on biochemistry of sample and spectra typical for: lipids, proteins, DNA. The aim of the study was to evaluate the biochemical and structural features of human colon cell lines based on Raman spectroscopy and imaging: normal cells CCD-18 Co, normal cells CCD-18 Co under oxidative stress conditions, normal cells CCD-18 Co at first treated by using tert-Butyl hydroperoxide and then supplemented by vitamin C in high concentration to show the protective role of vitamin C in micromolar concentrations against ROS by spectroscopic methods. Raman data obtained for normal cells injured by ROS were compared with spectra typical for cancerous cells. Statistically assisted analysis has shown that normal, ROS injured and cancerous human colon cells can be distinguished based on their unique vibrational properties. The research carried out proves that label-free Raman spectroscopy may play an important role in clinical diagnostics differentiation of normal and cancerous colon cells and may be a source of intraoperative information supporting histopathological analysis.