Many proteomics studies are limited to the identification of only the most abundant proteins in a sample due to the high sample complexity in most proteomes. We have here addressed this problem by prefractionation of human blood samples using microchromatography. We show that our approach resulted in high-stringency tryptic peptides identified by LC-ESI-MS/MS. Serum proteins were fractionated by batch and stepwise preparative chromatography using various types of chromatography resins (propyl sulfate, quaternary amine, diethylaminoethanol, cibachron blue, phenol Sepharose, carboxy methyl sepharose, hydroxyl apatite, heparin, concanavalin A and protein G) that were compared. The efficacy of sample fractionation was determined by protein assays, electrophoresis, and mass spectrometry. Tryptic peptides were separated by C18 liquid chromatography with electrospray ionization via metal needle at 2 microL/min with ion trap tandem mass spectrometry. The MS/MS spectra were correlated to some 4396 distinct sequences of the human forward RefSeq by X!TANDEM. Of these, 61% have been detected by other algorithms, but 3219 (73%) were never previously reported from blood by X!TANDEM. The use of a simple apparatus for making gravity microchromatography columns that permits the rapid side-by-side fractionation of many serum samples is described. Disposable microcolumns rapidly prepared blood samples for LC ESI-MS/MS that detected both tissue and cell leakage proteins known to exist in the approximately 1 ng/mL range and some circulating receptor sequences. Our results demonstrate that the depletion of albumin or IgG was not necessary prior to LC-MS/MS and that multiple forms of protein chromatography will be useful for complete identification of blood proteins.