AbstractRaman micro‐spectroscopy was used to discriminate between different types of bone cells commonly used in tissue engineering of bone, with the aim of developing a method of phenotypic identification and classification. Three types of bone cells were analysed: human primary osteoblasts (HOB), retroviral transfected human alveolar bone cells with SV40 large T antigen (SV40 AB), and osteoblast‐like human osteosarcoma derived MG63 cell line. Unsupervised principal component analysis (PCA) and linear discriminant analysis (LDA) of the Raman spectra succeeded in discriminating the osteosarcoma derived MG63 cells from the non‐tumour cells (HOB and SV40 AB). No significant differences were observed between the Raman spectra of the HOB and SV40 AB cells, confirming the biochemical similarities between the two cell types. Difference spectra between tumour and non‐tumour cells suggested that the spectral discrimination is based on the fact that MG63 osteosarcoma derived cells are characterised by lower concentrations of nucleic acids and higher relative concentrations of proteins compared to the non‐tumour bone cells. A supervised classification model (LDA) was built and showed high cross‐validation sensitivity (100%) and specificity (95%) for discriminating the MG63 cells and the non‐tumour cells, with 96% of the cells being correctly classified either as tumour or non‐tumour derived cells. This study proves the feasibility of using Raman spectroscopy to identify in situ phenotypic differences in living cells. © 2004 Wiley‐Liss, Inc.