A challenge of pediatric research is the limited ability to obtain tissue samples from small patients. To confront this problem, blood biomarkers can be used as surrogate markers of disease processes and aid in patient monitoring and disease detection. Furthermore, proteomic analysis of plasma samples is one approach for large-scale discovery of disease biomarkers. This study examined the use of plasma for disease process biomarkers in pediatric patients undergoing cardiopulmonary bypass (CPB) surgery. Proteomic studies of plasma are limited by the presence of a few high abundance proteins that mask the presence of lower abundance proteins of interest. Plasma immunoaffinity depletion (removing 6 of the highest abundance proteins of little pathological importance) increases sensitivity of detection for proteins such as those related to inflammation, remodeling, and damage. Using two-dimensional in-gel fluorescence electrophoresis, changes in the expression levels of proteins that occur as a result of CPB can be identified. In the present study, plasma depletion removed 83% of the plasma protein mass, allowing approximately 1400 spots to be observed by two-dimensional in-gel fluorescence electrophoresis. Of the detected spots, 79 (5.7%) were altered by CPB. These data illuminate the strength of plasma proteomics in identification of candidate biomarkers of CPB-associated disease processes.