A major limiting factor in stem cell therapy is the accurate identification of the differentiation state of cells destined for transplantation. This study aimed to evaluate the potential of synchrotron radiation Fourier transform infrared (SR-FTIR) microspectroscopy as a novel technique to probe the differentiation state of human mesenchymal stem cells (hMSCs) to chondrocytes over a period of 7, 14 and 21 days of induction. The chondrogenic markers were determined using reverse transcription polymerase chain reaction, histology and immunohistochemistry. The changes of average spectra located near 1338-1230 and 1175-960 cm(-1) indicated increased levels of collagen and aggrecan, respectively, in chondrocyte-induced hMSCs compared with control cells. Classification of independent test spectra using partial least squares discriminant analysis (PLS-DA) could distinguish control and chondrocyte-induced cells with 100 accuracy. We conclude that the SR-FTIR microspectroscopy technique is sensitive for monitoring the differentiation state of stem cells under chondrogenic induction particularly at an early stage. It provides biochemical information that is complimentary to that obtained from conventional techniques, and may give more unambiguous results particularly at the very early stage of cellular differentiation. In addition, the spectroscopic approach is more straightforward, non-destructive and requires less sample preparation compared with the conventional methodologies.