With advancements in the analytical technologies and methodologies in proteomics, there is great interest in biomarker discovery in biofluids such as serum and plasma. Current hypotheses suggest that the low molecular weight (LMW) serum proteome possesses an archive of clipped and cleaved protein fragments that may provide insight into disease development. Though these biofluids represent attractive samples from which new and more accurate disease biomarkers may be found, the intrinsic person-to-person variability in these samples complicates their discovery. Mice are one of the most extensively used animal models for studying human disease because they represent a highly controllable experimental model system. In this study, the LMW serum proteome was compared between xenografted tumor-bearing mice and control mice by differential labeling utilizing trypsin-mediated incorporation of the stable isotope of oxygen, 18O. The digestates were combined, fractionated by strong cation exchange chromatography, and analyzed by nanoflow reversed-phase liquid chromatography coupled online with tandem mass spectrometry, resulting in the identification of 6003 proteins identified by at least a single, fully tryptic peptide. Almost 1650 proteins were identified and quantitated by two or more fully tryptic peptides. The methodology adopted in this work provides the means for future quantitative measurements in comparative animal models of disease and in human disease cohorts.