Protein phosphorylation is a reversible posttranslational modification essential for intracellular signaling in cells. Disruptions in these processes are fundamental to cancerCancer development, as imbalances in phosphorylation can lead to aberrant signaling pathways driving tumor initiation, progression, and metastasis. Analyzing phosphorylation patterns in cancer cells can reveal critical signaling nodes and potential therapeutic targets. Reliable assessment of protein phosphorylation is crucial, and techniques such as phosphate-affinity SDS-PAGE, Western blotting, phospho-specific ELISA, mass spectrometry, and phospho-arrays are employed. This chapter focuses on phosphoflow technology, a flow cytometry-based assay, advantageous for its ability to analyze multiple cell populations and phosphorylation events simultaneously with minimal sample sizes. This method combines the principles of flow cytometry with specific antibodies that recognize phosphorylated epitopes on proteins. Phosphoflow allows to analyze multiple phosphorylated proteins simultaneously within a heterogeneous cell population without the need for cell purification. It is particularly useful for studying intracellular signaling pathways in various cell types, including those from clinical samples.