NK cells are effector cells of the innate immune system that are thought to play key roles in fighting viral infections and in tumor immune surveillance and eradication. It is shown that the effector functions of NK cells in patients with hematological malignancies are impaired and a correlative study have demonstrated a relationship between low NK cell cytotoxicity in blood and increased risk of malignancy. Several mechanisms explaining these defects have been proposed, amongst them down-regulation of NK cell surface receptors and defects in integration of intracellular signalling pathways. The aim of this study was to establish a protocol for phospho-flow cytometry to be used on frozen samples of PBMC and then utilize this protocol in the investigation of intracellular signal transduction in NK cells from pediatric leukemia patients. The protocol outlined in this paper has proven succesful in preparing frozen PBMC for analysis using phospho-flow cytometry. We have demonstrated that NK cells can be stimulated using anti-CD16 antibodies, stained with antibodies against intracellular phosphoepitopes and then permeabilized in order to evaluate intracellular phosphorylation states. This study includes four pediatric leukemia patients, each diagnosed with either AML, B-ALL or T-ALL. Though the patient population is small, our data does indeed suggest that there might be a decreased level of intracellular phosphorylation in CD16 stimulated NK cells in certain pediatric leukemia patients in comparison to healthy controls, thus supporting the existing evidence that NK cell functions are defective in these patients.