Raman spectroscopy is an advanced chemical analytical technique that has gained significant interest in cancer research, in particular early detection and monitoring of cancer, with added advantages of non-invasive and real-time diagnosis. Recently, studies have shown its sensitivity to monitor chemical changes during cancer progression. This information will lead to identification of chemical markers (molecular fingerprints of chemical composition) that can be used as biological markers. In this study, we used a tumor spheroid model that mimics the characteristics of a non-vascular in vitro tumor model, we used a combination of Raman and multivariate approach to identify chemical changes associated with normal proliferating, hypoxic and necrotic regions of T-47D human breast cancer spheroid model. The results provide evidence that lipids, amide I, III and nucleic acid contents differ significantly in normal, hypoxic and necrotic regions. Principal component analysis loading plots has suggested that normal proliferating region separated with low amide I and high-tryptophan content compared to hypoxic and necrotic regions. These differences observed in three regions might be useful in identification of new spectral markers associated stress faced by each region progressing toward necrosis.