ATM is a serine/threonine protein kinase that is responsible for initiation of DNA repair of double-stranded breaks and is a therapeutic target in cancer. A lack of analytically robust and multiplexed assays has hampered mechanistic studies of action and determination of optimal, robust pharmacodynamic biomarkers for clinical development of new therapies targeting ATM. Targeted mass spectrometry-based assays have been shown to be capable of enabling quantitative phosphosignaling studies and identification of novel phosphorylation sites. To identify new pharmacodynamic markers of ATM inhibition and expand the capabilities of targeted proteomics for quantitative profiling of the DNA damage response, we developed and fully analytically characterized a 51-plex assay quantifying protein expression and post-translational modification (phosphorylation) following induction of DNA damage. The linear range was over 3 orders of magnitude, the median inter-assay variability was 11% CV, and the assay is capable of being used in conjunction with other multiple reaction monitoring-based multiplexed assay panels. Proof-of-principle studies quantify signaling following DNA damage (ionizing radiation) in immortalized cell lines and primary human cells, identifying NUMA1 pS395 as a PD marker for ATM inhibition following DNA damage. Furthermore, the study shows the utility of using a quantitative multiplexed assay for studying cellular signaling dynamics, and the potential application to pharmacodynamic and mechanism of action studies, including development of new pharmacodynamic markers for clinical application.