In many studies, particularly in the field of systems biology, it is essential that identical protein sets are precisely quantified in multiple samples such as those representing differentially perturbed cell states. The high degree of reproducibility required for such experiments has not been achieved by classical mass spectrometry-based proteomic methods. In this manuscript we describe the implementation of a targeted quantitative approach by which pre-determined protein sets are first identified and subsequently quantified at high sensitivity reliably in multiple samples. This approach consists of three steps: First, the proteome is extensively mapped out by multidimensional fractionation and tandem mass spectrometry, and the data generated is assembled in the PeptideAtlas database. Second, based on this proteome map, peptides uniquely identifying the proteins of interest, proteotypic peptides (PTP’s), are selected and multiple reaction monitoring (MRM) transitions are established and validated by MS2 spectrum acquisition. This process of peptide selection, transition selection and validation is supported by a suite of software tools, TIQAM, described in this manuscript. Third, the selected target protein set is quantified in multiple samples by MRM. Applying this approach we were able to reliably quantify low abundant virulence factors from cultures of the human pathogen Streptococcus pyogenes exposed to increasing amounts of plasma. The resulting quantitative protein patterns enabled us to clearly define the subset of virulence proteins that is regulated upon plasma exposure.