A label-free semi-quantitative peptide feature profiling method was developed in response to challenges associated with analysis of two-dimensional liquid chromatography-tandem mass spectrometry data. One hundred twenty human sera (49 from invasive breast carcinoma patients, 26 from non-invasive breast carcinoma patients, 35 from benign breast disease patients, and 10 from normal controls) were repeatedly analyzed using a standardized two-dimensional liquid chromatography-mass spectrometry method. Data were extracted using the novel semi-quantitative peptide feature profiling method, which is based on comparisons of normalized relative ion intensities. Hierarchical cluster analyses and principle component analyses were used to evaluate the predicative capability of the extracted data, and results were promising. Extracted data was also randomly assigned to either a training group (65%) or to a test group (35%) for artificial neural network modeling. Models best identified invasive breast carcinomas (212 predictions, 94% accurate) and benign non-neoplastic breast disease (96 predictions, 81.3% accurate). These results suggest that, after further development, the novel method may be useful for large-scale clinical proteomic profiling.