Bar adsorptive microextraction combined with liquid desorption followed by high performance liquid chromatography with diode array detection (BAµE-LD/HPLC-DAD) is proposed for the determination of the psychoactive alkaloid mitragynine (MG) in human urine matrices. By using a modified N-vinylpyrrolidone polymer (P2) sorbent phase, high selectivity and efficiency is achieved. Assays performed by BAµE(P2)-LD/HPLC-DAD on 25 mL water samples spiked at the 8.0 µg L−1 level yielded average recoveries around 100% of MG, under optimized experimental conditions. The analytical performance showed good precision (RSD