The dried blood spot (DBS) methodology provides a minimally invasive approach to sample collection and enables room-temperature storage for most analytes. DBS samples have successfully been analyzed by liquid chromatography multiple reaction monitoring mass spectrometry (LC/MRM-MS) to quantify a large range of small molecule biomarkers and drugs; however, this strategy has only recently been explored for MS-based proteomics applications. Here we report the development of a highly multiplexed MRM assay to quantify endogenous proteins in human DBS samples. This assay uses matching stable isotope-labeled standard peptides for precise, relative quantification, and standard curves to characterize the analytical performance. A total of 169 peptides, corresponding to 97 proteins, were quantified in the final assay with an average linear dynamic range of 207-fold and an average R2 value of 0.987. The total range of this assay spanned almost 5 orders of magnitude from serum albumin (P02768) at 18.0 mg/ml down to cholinesterase (P06276) at 190 ng/ml. The average intra-assay and inter-assay precision for 6 biological samples ranged from 6.1–7.5% CV and 9.5–11.0% CV, respectively. The majority of peptide targets were stable after 154 days at storage temperatures from −20 °C to 37 °C. Furthermore, protein concentration ratios between matching DBS and whole blood samples were largely constant (<20% CV) across six biological samples. This assay represents the highest multiplexing yet achieved for targeted protein quantification in DBS samples and is suitable for biomedical research applications.