Sepsis is a systemic, often fatal inflammatory response whose biochemical pathways are not fully understood and with no single biomarker capable of its reliable prediction. Increased interest in protein profiling to reveal fundamental biochemical events as well as disease diagnosis has grown considerably, largely due to advances in mass spectrometry and related front-end technologies. In this study, patients with sepsis and systemic inflammatory response syndrome (SIRS) were examined using plasma protein profiling following immunodepletion treatment to remove the most abundant proteins, serum albumin, transferrin, haptoglobin, anti-trypsin, IgG, and IgA. These proteins cause significant signal suppression, and their removal allows for lower abundance proteins to be examined through improved ion signal. Analyses after immunodepletion were performed using 3-dimensional reverse phase/strong cation exchange/reverse phase liquid chromatography with electrospray ion trap mass spectrometry (3D LC-MS/MS) and spectrum counting for comparative quantitation. The results revealed a major theme in immune system activity, including activation of the complement and coagulation pathways. Additionally, lipid transport may prove to be important in distinguishing sepsis from SIRS. Specifically, significant multi-fold changes were observed in 10 proteins and are now being investigated for the early diagnosis of sepsis.