Abstract Introduction. Acute Myeloid Leukemia (AML) is the most common acute Leukemia among adults with a poor overall prognosis. With over 20,000 cases in the US alone, about a third of AML patients pertain to an important oncogenic driver composed of internal tandem duplication (ITD) somatic mutations in exons 14 and 15 of the FMS-like tyrosine kinase 3 (FLT3) gene. Detecting the presence of measurable residual disease in patients with AML who are in morphologic remission has been shown to be a powerful predictor of eventual relapse. Yet, despite its importance, the ability to detect FLT3-ITD mutations is hampered by the limited sensitivity of conventional PCR-based assays in general and in Next Generation Sequencing-based assays, in part due to the difficulty to align ITD-containing reads to the reference genome. Methods. In order to facilitate the analysis of AML samples, we developed FLT3-Explorer, a comprehensive bioinformatics pipeline composed of open source software, combined with in-house algorithms to accurately detect low levels of FLT3-ITD mutations. The input of the pipeline is unmapped reads in fastq format. The pipeline then searches for either ITD sequences previously observed in our extensive database of historical data, or a de-novo detection procedure is used to determine mutation sequences of variable lengths within the FLT3 gene. To assess the performance of FLT3-Explorer, we considered a cohort of clinical samples from bone marrow and peripheral blood, as well as cell lines. For sample collection, genomic DNA was isolated, and polymerase chain reaction was performed using primers flanking exons 13 and 15 to amplify the FLT3 gene. DNA was then sequenced using NGS Illumina sequencing. Results were validated using fragment size analysis by Capillary Electrophoresis assays. Also, to determine the limits of detection, various dilutions were performed on cell lines. Furthermore, to assess the performance of our pipeline, we performed a comparison with other FLT3-ITD programs publicly available. Summary. Results from our analysis showed that our method identifies FLT3-ITD mutations of variant allele fraction as low as 0.003%. Also, we found that our pipeline can detect as many FLT3-ITD mutants as other methods, or more, when the mutant signal is considerably low. Conclusions. FLT3-Explorer constitutes a powerful solution for low FLT3-ITD detection signal of mutant sequences, of diverse lengths, that can be used on FLT3-ITD AML patients to track disease progression and relapse. Citation Format: Christian Laing, Wenge Shi, Reinhold Pollner. FLT3-Explorer: A bioinformatics pipeline for detecting low signal in FLT3 internal tandem duplications in acute myeloid leukemia abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2475.