Dysregulation of cytokine signaling pathways is associated with benign and malignant hematologic disorders. Improvements in therapy rely on understanding the biology of the pathways and the proteins involved. Studying these pathways in patient samples is challenging as samples are difficult to obtain, contain fewer cells, and are heterogeneous in nature. To address some of these difficulties, we have utilized the technique of microcapillary electrophoresis. Using the NanoPro 1000 system (ProteinSimple) which is built on an automated, capillary-based immunoassay platform, we have developed rapid and quantitative assays for specific proteins from relatively small sample sizes. The NanoPro provides precise and quantitative data of the phosphorylation states of a specific protein of interest. We describe our experience with NanoPro assay development and optimization with specific application toward understanding aberrant cytokine signaling in human leukemia cells.