The ability to effectively monitor the behaviour of pluripotent stem cells and their differentiation is key to their use in basic and clinical research. Molecules expressed in particular cell types can be used to report the status of cell differentiation and is a recognised means of assessing the behaviour of cell cultures. There are currently few useful markers of stem cells and there is no rapid way to accurately determine their level of expression. In this study, we describe for the first time the potential of surface enhanced laser desorption/ionisation time-of-flight mass spectrometry (SELDI-TOF-MS) to identify novel biomarkers of human pluripotent embryonal carcinoma stem cells and their differentiated derivatives. This approach allows the rapid and sensitive screening of cell samples without the need to purify the specimen prior to analysis. The identification of proteins expressed in specific cell populations will provide valuable tools for monitoring cellular development.