Conatumumab is a monoclonal antibody specific for death receptor 5 (DR5) that activates caspases leading to DNA fragmentation and tumor-cell death. Like other Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL) receptor therapies, conatumumab is currently being evaluated in clinical trials across a variety of tumor types. However, molecular evidence of on-target drug activity in tumors is often an elusive goal for clinical investigation. Here we evaluated a translational approach using a relevant biopsy method, fine needle aspirates (FNAs), to study the on-target pharmacodynamics of conatumumab pre-clinically. As detected by laser scanning cytometry, drug-induced caspase-3 activation in FNA biopsies of Colo205 xenografts correlated well with activated caspase-3 in conventional section-based samples. Furthermore, in tumor-bearing mice, surrogate assays of serum caspase-3/7 activity and serum drug exposure correlated with in situ caspase-3 activation. We found that one advantage of FNA sampling over other sampling techniques was the ability to measure caspase activity on a per cell basis using DNA content information. To adapt the utility of FNAs for measuring pharmacodynamic markers in humans, detection of activated caspase-3 was multiplexed with EpCAM to characterize mock and clinical FNAs from colorectal and nonsmall cell lung cancer patients. These data suggest that FNA sampling is a practical method to cytometrically evaluate tumors for pharmacological impact in a clinical setting.