Surface-enhanced laser desorption time of flight mass spectrometry (SELDI-TOF-MS) is an important proteomic technology that is immediately available for the high throughput analysis of complex protein samples. Over the last few years, several studies have demonstrated that comparative protein profiling using SELDI-TOF-MS breaks new ground in diagnostic protein analysis particularly with regard to the identification of novel biomarkers. Importantly, researchers have acquired a better understanding also of the limitations of this technology and various pitfalls in biomarker discovery. Bearing these in mind, great emphasis must be placed on the development of rigorous standards and quality control procedures for the pre-analytical as well as the analytical phase and subsequent bioinformatics applied to analysis of the data. To avoid the risk of false-significant results studies must be designed carefully and control groups accurately selected. In addition, appropriate tools, already established for analysis of highly complex microarray data, need to be applied to protein profiling data. To validate the significance of any candidate biomarker derived from pilot studies in appropriately designed prospective multi-center studies is mandatory; reproducibility of the clinical results must be shown over time and in different diagnostic settings. SELDI-TOF-MS-based studies that are in compliance with these requirements are now required; only a few have been published so far. In the meantime, further evaluation and optimization of both technique and marker validation strategies are called for before MS-based proteomic algorithms can be translated into routine laboratory testing.