<p>The first nanotechnology-based innovative analytical strategy by means of dispersive solid-phase extraction (DSPE) as a necessary sample treatment approach for the simultaneous determination of revumenib and venetoclax in complex biological matrices&#xa0;such as human blood serum prior to high-performance liquid chromatography hyphenated to diode-array detector (HPLC-DAD) is reported. Several carbon nanotubes were rigorously evaluated as DSPE sorbents to obtain an adequate extraction and preconcentration of these chemotherapeutic agents. The best findings were 8 mg of pristine multi-walled carbon nanotubes (MWCNTs) in borate buffer (pH 10; 10 mM). Under optimized chromatographic conditions, the detection, identification, and determination of both anticancer agents and trazodone (internal standard) were done in less than 8 min of analysis. The HPLC-DAD separation was carried out in a C18 reversed-phase column with a mobile phase including ammonium acetate (pH 7; 10 mM) and methanol throughout a gradient elution mode with a sample flow rate of 0.9 mL min^-1 and a column temperature of 26°C. The preconcentration factor achieved was 1.7 so the limits of detection and quantification were 0.8 μg L^-1 and 2.6 μg L^-1 for revumenib and 0.7 μg L^-1 and 2.3 μg L^-1 for venetoclax in serum samples. The proposed strategy stands up as an interesting approach for therapeutic drug monitoring in AML patients moving away from a “one-size-fits-all” approach.</p> Graphical Abstract <p></p>