Multiple Reaction Monitoring (MRM) is a highly sensitive and selective mass spectrometry technique for peptide quantification in proteomics. The development of an MRM assay method is critical for highly rapid, accurate, and reproducible measurements. We developed an automated workflow to generate optimal MRM transitions based on empirical synthetic peptide libraries produced by flow injection analysis using in-house developed software called MRMOptimizer. Furthermore, the evaluation of alternative techniques to MRM, such as multiple reaction monitoring cubed (MRM³) and differential ion mobility mass spectrometry (DMS), in terms of selectivity and sensitivity was an essential part of this thesis. Finally, we applied our optimized MRM assay method to investigate human monocyte-derived dendritic cell (MDDC) protein expression during the early HIV-1 infection state.