Identification of unknown proteins subsequent to a mass spectrometry signal is still a serious obstacle in the discovery of relevant biomarkers of diagnostic interest. In this report the evaluation of a rational process under optimized conditions is described for three unknown proteins representing important targets in their field of investigation. The process, involving few dozens of chromatographic sorbents and two buffers, allowed identifying prothrombin fragment 1, a minor glycoprotein of human serum with inhibitory activity associated with pathogenesis of calcium oxalate stones. The same technology demonstrated its efficiency for the separation of a recombinantly expressed yeast transcription factor in Escherichia coli with subsequent formal identification. In addition, a DNA-binding protein from the gastric pathogen Helicobacter pylori has been separated by the same technology and formally identified. The reported data show that the method is reliable and easily applicable to a large variety of cases with a standardized approach. Identity coverage and relative abundance after purification and removal of critical protein impurities are reported. Examples of protein isolation/identification are described, namely PTF1, recombinant YAP-1 transcription factor from E. coli and DNA-binding protein HU from H. pylori. Isolated proteins were pure enough for the purpose of formal identification by either peptide mass fingerprinting or sequencing.