Phosphorylation of proteins plays a pivotal role in signal transduction processes, and it is a key regulator of many biological cell functions. Various strategies have been proposed for the study of phosphoproteome; most of them require a multi-step analysis and sophisticated equipment. Here we describe the two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) analysis of PBMC phosphoproteome using as preliminary enrichment step a simple phosphoprotein isolation by lanthanum chloride. This strategy can be most certainly applied to study the phosphoproteome of CTLs isolated from PBMCs. The phosphoproteome analysis of PBMCs, as well as of CTLs, may help to reveal the signaling pathways essential to their biological role in health and disease.