Raman spectroscopy is employed to determine the suitability of the U20S osteoblast-like cell line for use as a model for human primary osteoblasts, with emphasis on the ability of these cell types to replicate their tissue of origin. It was found that both cell types demonstrated early stage mineral deposition that followed significantly different growth patterns. Analysis of the growth pattern and spectral data from primary cells revealed increasing bone quality ratios and a high crystallinity, consistent with previous reports. Conversely the investigation of the U20S osteoblast-like cell line provided evidence of dense multilayered mineralised regions that corresponded more closely to native bone in terms of its crystallinity and bone quality ratios. This finding contradicts previous reports on U20S osteoblast-like cells which have consistently described them as non-osteoinductive when cultured in various conditions on a number of substrates. This work demonstrates the successful application of Raman spectroscopy combined with biological and multivariate analysis for the investigation of osteoblast-like U20S cells and human primary osteoblasts, specifically with focus on the osteoinductive ability of the osteoblast-like cell line and the comparative differences in relation to the primary osteoblasts.