Abnormal expression of key signaling molecules and defective functions of T lymphocytes play a significant role in the pathogenesis of systemic lupus erythematosus (SLE). T cell receptor (TCR/CD3)-mediated stimulation of SLE T cells show increased protein tyrosine phosphorylation of cellular proteins with faster kinetics, heightened calcium flux response, and decreased IL-2 production. The molecular mechanisms of T cell signaling abnormalities in SLE T cells are complex. Current research has been directed towards investigating various factors that contribute to abnormal tyrosine phosphorylation, intracellular calcium response, and cytokine production. Central to this dysfunction is the aberrant expression and function of the TCR/CD3ζ chain. Latest developments suggest multiple explanations are involved, including altered receptor structure, supramolecular assembly, modulation of membrane clustering, aberrant cellular distribution, and pre-compartmentalization with lipid-rafts. The methods and protocols described here pertaining to T cell signaling abnormalities in SLE T cells are optimized in many ways and are derived by the combined task and continuous efforts of many researchers in the lab over a long period of time. These simplified protocols can be readily applied to study T cell signaling abnormalities in SLE to identify the genetic, molecular, and biochemical factors contributing to aberrant immune cell function and unravel the pathophysiology of SLE.