There is a growing body of evidence showing that optical spectroscopy has the potential to be a useful in vivo diagnostic tool. Yet, so far there is no definitive cellular and biochemical understanding for the differences seen in the spectra from different tissue categories and disease states. In this study, we examine the use of organotypic raft cultures as an in vitro model of in vivo tissue conditions in an attempt to overcome some of the limitations of previously used methods. Organotypic raft cultures resembling normal and dysplastic epithelial cervical tissue were constructed and grown at an air-liquid interface for 2 weeks. Raman spectra of normal as well as dysplastic raft cultures were measured and compared with in vivo spectra from the corresponding tissue type. Histologic comparisons ensured that the raft cultures had similar structure and morphology to the corresponding intact tissue types. Raman spectra were also acquired from different layers of tissue. Spectral comparisons show that the Raman spectra of the raft cultures are similar to the spectra acquired from the cervix in vivo for both normal and dysplastic tissues. These results show that organotypic raft cultures are an effective and useful tool for the cellular and biochemical analysis of tissue spectroscopy.