Identification of druggable mutations in Acute Myeloid Leukemia (AML), such as FLT3-ITD, has led to the development of targeted therapies capable of out-performing cytotoxic chemotherapy. Despite early survival benefits, most patients with FLT3-ITD AML on trial with the FLT3 inhibitor (FLT3i) gilteritinib still develop treatment resistance. Critical readout of response to FLT3i as measured by phospho-signaling is missing for the majority of these patients. Here, we validated an innovative approach to obtaining these signatures by leveraging the Smart BufferTM preservation system for peripheral blood (PB) specimens integrated with phospho-mass cytometry as part of the ongoing BeatAML Trial.