Deleterious mutations in genes involved in the Fas apoptosis pathway lead to Autoimmune Lymphoproliferative Syndrome (ALPS). Demonstration of an apoptosis defect is critical for the diagnosis and study of ALPS. The traditional in vitro apoptosis assay, however, requires a week of experimental procedures. Here, we show that defects in Fas-induced apoptosis in PBMCs can be evaluated directly ex vivo using multicolor flow cytometry to analyze the apoptosis of effector memory T cells, a Fas-sensitive subset of PBMCs. This method allowed us to sensitively quantify defective apoptosis in ALPS patients within a few hours. Some ALPS patients (ALPS-sFAS) without germline mutations have somatic mutations in Fas specifically in double-negative αβ T cells (DNTs), an unusual lymphocyte population that is characteristically expanded in ALPS. Since DNTs have been notoriously difficult to culture, defective apoptosis has not been previously demonstrated for ALPS-sFAS patients. Using our novel ex vivo apoptosis assay, we measured Fas-induced apoptosis of DNTs for the first time and found that ALPS-sFAS patients had significant apoptosis defects in these cells compared to healthy controls. Hence, this rapid apoptosis assay can expedite the diagnosis of new ALPS patients, including those with somatic mutations, and facilitate clinical and molecular investigation of these diseases.