In response to the alarming rates of zolpidem abuse, this study presents a pioneering approach utilizing molecularly imprinted nanoparticles (MIPs) in conjunction with high‐performance liquid chromatography (HPLC) for the selective detection of zolpidem. The rational choice of zolpidem as the target analyte stems from its widespread misuse and clinical significance. The MIPs were prepared using the precipitation polymerization method, resulting in monodispersed nanoparticles with a size of approximately 258.8 nm. Fourier Transform Infrared (FTIR) analysis confirmed the successful incorporation of zolpidem into the MIPs, designed with tailored cavities for zolpidem recognition. Moreover, incorporating the bathochromic shift phenomenon reaffirms the successful imprinting procedure. The rebinding studies showed that MIPs were highly efficient in adsorbing zolpidem from the solvent, with more than 95 % of the drug recovered after multiple cycles. The selectivity study demonstrated the MIPs′ preference for zolpidem over another drug, diazepam. The MIPs′ successful separation of zolpidem from spiked human plasma samples further validates their potential for practical applications, such as clinical toxicology. This research showcases the potential of MIPs coupled with HPLC as a powerful tool for accurate zolpidem quantification, holding great promise for combating drug abuse and advancing analytical methodologies for pharmaceutical analysis.