Context Detection of measurable residual disease after therapy is an important predictor of outcome in acute myeloid leukemia. Objective To investigate the feasibility of using next-generation sequencing (NGS) in the diagnostic laboratory to perform quantitative NPM1 mutation assessment using ultradeep (approximately 300 000×-500 000×) sequencing (NGS-q NPM1) as a method of assessing residual disease burden in patients with acute myeloid leukemia. Design A flexible NGS-based assay for the detection and quantitation of NPM1 mutations was developed by polymerase chain reaction amplification of target DNA sequences, sequencing on an Illumina (San Diego, California) MiSeq, and analyzing data with an in-house-designed bioinformatic pipeline. NGS-q NPM1 was compared with current NPM1 quantitation methods (real-time quantitative-polymerase chain reaction and multiparameter flow cytometry). Results The NGS-q NPM1 assay had a sensitivity of between 10-4 and 10-5 and showed high concordance and correlation with reference methodologies. Moreover, the NGS-q NPM1 assay was able to be integrated into the laboratory's existing, targeted amplicon-based sequencing workflow. Conclusions An NGS-based, quantitative NPM1-mutation assessment can be used to monitor patients with acute myeloid leukemia, and it has some practical advantages over existing modalities.