Serum protein profiling is a promising approach for classification of cancer versus non-cancer samples. The objective of our study was to assess the feasibility of mass spectrometry based protein profiling for the discrimination of colorectal cancer (CRC) patients from healthy individuals. In a randomized block design, pre-operative serum samples obtained from 66 colorectal cancer patients and 50 controls were used to generate MALDI-TOF protein profiles. After pre-processing of the spectra, linear discriminant analysis with double cross-validation was used to classify protein profiles. A total recognition rate (92.6%), sensitivity (95.2%) and specificity (90.0%) for the detection of CRC were shown. The area under the curve of the classifier was 97.3%, and demonstrated the high, significant separation power of the classifier. Double cross-validation shows that classification can be attributed to information in the protein profile. Although preliminary, the high sensitivity and specificity indicate the potential usefulness of serum protein profiles for the detection of colorectal cancer.