In this study, a novel approach is proposed for determination of methylphenidate in biological fluids. In this method molecularly imprinted solid-phase extraction (MISPE), as the sample extraction technique, combined with high-performance liquid chromatography (HPLC) is used. The water-compatible molecularly imprinted polymers (MIPs) were prepared using methacrylic acid as functional monomer, ethylene glycol dimethacrylate as cross-linker, Hexane as porogen and methylphenidate as template molecule. Extraction of methylphenidate from human serum was carried out using a novel imprinted polymer as the solid-phase extraction (SPE). Various parameters affecting the extraction efficiency of the polymer were evaluated. Also, the optimal conditions for the MIP cartridges were studied. The limit of detection (LOD) and limit of quantification (LOQ) for methylphenidate in serum samples were 1.3 and 10 ng mL -1 , respectively. The recoveries for serum samples were higher than 92%.