Mutations in the gene encoding nucleophosmin (NPM1) carry a prognostic value for patients with acute myeloid leukemia (AML). Various techniques are currently being used to detect these mutations in routine molecular diagnostics. Incorporation of accurate NPM1 mutation detection on a gene expression platform would enable simultaneous detection with various other expression biomarkers. Here we present an array-based mutation detection using custom probes for NPM1 WT mRNA and NPM1 type A, B, and D mutant mRNA. This method was 100% accurate on a training cohort of 505 newly diagnosed unselected AML cases. Validation on an independent cohort of 143 normal-karyotype AML cases revealed no false-negative results, and one false positive (sensitivity 100.0% and specificity 98.7%). Based on this, we conclude that this method provides a reliable method for NPM1 mutation detection. The method can be applied to other genes/mutations as long as the mutant alleles are sufficiently highly expressed.