Surface enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) is a widely used technology platform for diagnostic biomarker discovery in tissue, plasma and serum. High-throughput and simplicity of experimental procedures have allowed this technology to become an important research tool for biomarker discovery during the last years. This review provides an overview of SELDI-TOF functionality, its advantages and drawbacks and gives a current literature overview of colorectal cancer based serum biomarker detection. Further improvements in instrumentation sensitivity and labelling chemistries will enable detection of novel, tissue-leakage biomarkers in serum. However, major emphasis should be given on subsequent identification of differentially observed protein peaks detected by SELDI-TOF. Clinical validation in large patient cohorts will then allow transferring novel biomarkers into clinical use.