Diagnosis of T-cell large granular lymphocytic leukemia (T-LGL) is often challenging because clinical and laboratory characteristics are overlapping with nonneoplastic conditions. Recently, mutation in the STAT3 gene has been identified as a recurrent genetic abnormality in T-LGL. STAT3 mutation, therefore, represents a promising marker in T-LGL diagnostics. We developed a new quantitative next-generation sequencing assay that allows sensitive analysis of the STAT3 gene. The assay was used to study the utility of STAT3 mutation analysis as a diagnostic tool in T-LGL. The study included 16 T-LGL patients. A total of 15 mutations, including 2 new mutations (G618R and K658R), were detected in 12 patients (75%), with mutation levels ranging from 2.5% to 45.6% mutation-positive alleles. Next-generation sequencing detected 50% more mutations than Sanger sequencing. Blood samples from 20 healthy blood donors all tested negative, thus demonstrating the specificity of the assay. The results also indicated that mutation levels in blood and bone marrow are not systematically different, and next-generation sequencing-based STAT3 mutation analysis represents a sensitive method for monitoring residual disease as demonstrated in a patient receiving pentostatin. We demonstrate the clinical relevance of next-generation sequencing-based STAT3 mutation analysis, which represents a sensitive and specific diagnostic marker in T-LGL that allows assessment of molecular residual disease, which may improve clinical decision making.