The use of proteomics technology during the development of a new process for plasma protein separation was demonstrated. In a two-step process, the two most abundant proteins, HSA and IgG, were removed in a first step of anion-exchange chromatography using a gel with very high capacity. Subsequently, two fractions containing medium and low abundance proteins were re-chromatographed on a smaller column with the same type of gel. Collected fractions were separated by SDS-PAGE and 2-D electrophoresis, and excised proteins were digested with trypsin and identified by LC-ESI-MS/MS. This proteomic analysis proved to be a useful method for detection of low abundance therapeutic proteins and potential harmful contaminants during process development. Based on this method, low abundance therapeutic proteins, such as vitamin-K-dependent clotting factors and inhibitors, could be identified as present in target fractions after chromatographic separation. In addition, the tracking of potentially dangerous impurities and designing proper steps for their removal are important outcomes when developing, refining or controlling a new fractionation schema. For the purpose of in-process control, in-solution digestion of complete fractions followed by protein identification with LC-ESI-MS/MS was demonstrated as a rapid and simple alternative to the entire analysis including 1-D or 2-D electrophoretic steps.