Multispectral assessment of cell autofluorescence gives a direct window into the molecular processes occurring within those cells. This can be used to non-invasively characterise and classify various cellular properties without requiring fixation, dyes or transformation. Human mesenchymal stem/stromal cells (MSCs) have great potential to contribute to regenerative medicine, especially with regards to autologous transplantation. However, this capacity is often limited by inherent properties of cell lines, which prevent their being sufficiently expanded after derivation for effective clinical application. The investigation of these properties requires numerous, time and labour-intensive assays. In this study we have used correlative microscopy based on multispectral images of cell autofluorescence then correlated to functional assays in order to construct multispectral signatures of numerous inherent cell characteristics. These included cell cycle status (indicating the proportion of cells undergoing cell division at a given time), cell 'age' (number of passages undergone, indicating capacity for further expansion), and β-galactosidase (a marker of senescence, indicating cells which can no longer divide). This study has established a single protocol, in place of multi-functional assays, to characterize the growth and differentiation capacity of hMSC lines using a non-invasive approach.