The use of protein biomarkers as surrogates for clinical endpoints requires extensive multilevel validation including development of robust and sensitive assays for precise measurement of protein concentration. Multiple reaction monitoring (MRM), is a well-established methodology that can be used for reproducible protein-concentration measurements in biological specimens collected via microsampling. The dried blood spot (DBS) microsampling technique can be performed non-invasively without the expertise of a phlebotomist, and can enhance analyte stability which facilitate the application of this technique in retrospective studies. Thus, precise, sensitive, and multiplexed methodologies for measuring protein concentrations in DBSs can be used for de novo biomarker discovery. To achieve this goal, MRM assays were developed for multiplexed concentration measurement of proteins in DBS. The lower limit of quantification (LLOQ) was found to have a median total coefficient of variation (CV) of 18% for 245 proteins, while the median LLOQ was 5 fmol of peptide injected on column, and the median inter-day CV over 4 days for measuring endogenous protein concentration was 8%. The majority (88%) of the assays displayed parallelism, while the peptide standards remained stable throughout the assay workflow and after exposure to multiple freeze-thaw cycles. For 190 proteins, the measured protein concentrations remained stable in DBS stored at ambient laboratory temperature for up to 2 months. Finally, the developed assays were used to measure the concentration ranges for 200 proteins in twenty same sex, same race and age matched individuals.