Coliform mastitis has been a primary focus of dairy cattle disease research due to staggering affiliated losses, severe systemic complications arising from host inflammatory response to lipopolysaccharide, and the poor response of coliform pathogens to antimicrobials. Reliable biomarkers are needed to evaluate the efficacy of adjunctive therapies for the treatment of inflammation associated with coliform mastitis, and to aid in the approval of new veterinary drugs. The aims of the current analyses were to utilize proteomic methodologies to evaluate protein expression in whey from cows with experimentally induced coliform mastitis, and to employ label-free quantification strategies to estimate changes in relative abundance of proteins identified in milk over the course of clinical infection. Two-dimensional gel electrophoresis followed by matrix-assisted laser desorption/ionization time-of-flight (MALDI- TOF) mass spectrometry (MS) resulted in the identification of complement factors, antimicrobial proteins, and acute phase proteins in mastitic milk. Analysis using liquid chromatography (LC) inline with electrospray ionization - quadrupole TOF tandem mass spectrometry (MS/MS) resulted primarily in the identification of abundant whey and casein proteins, and the transient detection of proteins related to host response. Nano-LC- nanospray-MS/MS using a linear ion trap, however, led to the robust discovery of over fifty inflammatory proteins in whey from mastitic milk, including the novel markers kininogen-2 and inter-alpha trypsin inhibitor heavy chain-4. Normalized spectral counts were compared to enzyme-linked immunosorbant assay (ELISA) for select proteins to assess the accuracy of the spectral count data. Similar expression patterns were detected using spectral counts and ELISA. Results indicate that proteomic methodologies can detect biomarkers of coliform mastitis in bovine milk during clinical infections, and that spectral counts are a viable means of evaluating relative changes in protein biomarkers of mastitis, including those for which no antibody currently exists.