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Abstract: A simple and highly sensitive method for simultaneous detection of anticancer drugs is developed by integrating the preconcentration and separation steps in a microfluidic device with an amperometric biosensor. An amperometric detection with dsDNA and cardiolipin modified screen printed electrodes are used for the detection of anticancer drugs at the end of separation channel. The preconcentration capacity is enhanced thoroughly using field amplified sample stacking and field amplified sample injection techniques. The experimental parameters affecting the analytical performances, such as pH, temperature, buffer concentration, water plug length, and detection potential are optimized. A reproducible response is observed during multiple injections of samples with a RSD <5%. The calibration plots are linear with the correlation coefficient between 0.9913 and 0.9982 over the range of 2–60 pM. The detection limits of four drugs are determined to be between 1.2 (±0.05) and 5.5 (±0.3) fM. The applicability of the device to the direct analysis of anticancer drugs is successfully demonstrated in a real spiked urine sample. Device was also examined for interference effect of common chemicals present in real samples. [Copyright &y& Elsevier]