<p>An on-chip electromembrane extraction integrated with solid-phase microextraction (EME-SPME) was developed for the determination and monitoring of anticancer drugs, including imatinib, irinotecan, and mitomycin C, in various biological fluids. The extraction device incorporated an electrospun polyacrylonitrile (PAN)/MOF-303 nanocomposite fiber, which functioned as the extraction phase. Simultaneous analyte migration under an applied electric field and adsorption onto the nanocomposite surface enabled efficient preconcentration within the compact microfluidic platform. Under optimized conditions, the proposed EME-SPME–HPLC–UV method exhibited low limits of detection ranging from 0.1 to 1.5 µg L^–1. Satisfactory linearity was achieved across the ranges 0.5–1000 ng mL^–1 for imatinib and 5–1000 ng mL^–1 for both irinotecan and mitomycin C, with coefficients of determination (R²) ≥ 0.9938. The method also demonstrated acceptable precision, with relative standard deviations (RSDs) ≤ 7.5%. The applicability of the system was investigated through the extraction of target analytes from human urine and plasma samples, yielding relative recoveries between 78 and 110%. These results highlight the potential of the developed EME-SPME platform as a sensitive, precise, and environmentally friendly technique for therapeutic drug monitoring of anticancer agents in complex biological matrices.</p> Graphical Abstract <p></p>