Abstract Background Rearrangements of mixed lineage leukemia gene (MLL) with 1 of >130 alternative partner genes is a recurrent cytogenetic finding in both acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) and is generally associated with a poor prognosis. Among the most common MLL rearrangement is t(4;11)(q21;q23), forming the MLL::AF4 (also known as KMT2A::AFF1) fusion gene. Unique among MLL rearrangements, MLL::AF4 is almost exclusively associated with pro-B cell ALL and is prototypical of infant ALL, where it carries a very poor prognosis. At least 10 different MLL-AF4 fusion transcripts have been found, due to break-points in different introns, some transcripts being more frequent in either adult or infant ALL. Fast and reliable diagnostic tests represent an important tool in clinical laboratories to detect these rearrangements and . In this context, this work aimed to validate a qualitative method to detect the main MLL::AF4 fusion isoforms. Methods We evaluated the performance of the Hs03043614_ft (e10e4, e9e4 and e9e5) and Hs03024412_ft (e11e4) inventoried TaqMan assays (Thermo Fisher Scientific). Total mRNA from negative and positive samples for each fusion was extracted by the TRIzol reagent according to the manufacturer’s instructions. All assays were performed using TaqMan RNA-to-Ct 1-Step kit (Thermo Fisher Scientific). The inventoried ABL1 assay (Thermo Fisher Scientific) was used as endogenous control. Using positive samples, negative samples and controls of each fusion were performed: sequence verification tests, standardization of RNA input, evaluation of reaction efficiency and determination of the detection limit (LOD). The performance of the assays was evaluated with ABL1 control separately from the fusions. Results Our results showed that both assays worked well for the detection of specific fusions. The best RNA input for the reactions was 200 ng. The efficiencies reactions were: a) with ABL1 in separate reactions: 92.6% and 110.0% for Hs03043614_ft and Hs03024412_ft assays, respectively; The literature recommends the limit of efficiency was between 90% and 110%. The LODs with ABL1 in separate reactions were 10−3 for both assays, respectively. There was no unspecific amplification in negative samples. Conclusion The TaqMan assays with RT one-step protocol evaluated are highly sensitive, fast, and reliable tools for detecting the most frequent MLL::AF4 fusion transcripts.