The immune system is shaped by the random generation of lymphocytes followed by apoptosis of self-reactive cells, a process termed negative selection. The survival of these pathogenic cells in the periphery can elicit autoreactivity. We describe the development of a biomarker assay for the detection of pathogenic subpopulations of lymphoid cells in adult non-obese diabetic (NOD) mice based on disease-specific alterations in spontaneous or triggered cell death. Utilizing improved methods of cell separations, two distinct lymphoid cell subpopulations with increased susceptibility to apoptosis were identified and quantified. A subpopulation of CD8+ T cells that constitutes approximately 3-7% of the total CD8+ T cell population underwent apoptosis on exposure to low concentrations of TNF-alpha. Such cells were exclusively detected only in NOD mice with histologic signs of active autoreactivity. The non-T cell compartment of NOD immune system, although resistant to TNF-alpha-induced apoptosis, contained a subpopulation of B cells with spontaneous death by culture alone. The refined detection of small numbers of lymphoid cell subsets with quantifiable differences in apoptosis provides a possible immune biomarker for monitoring disease activity or treatment interventions.