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Human performance enhancing drugs (PEDs), frequently used in sport competitions, are strictly prohibited by the World Anti-Doping Agency (WADA). Biological samples collected from athletes and regular patients are continuously tested regarding the identification and/or quantification of the banned substances. Current work is focused on the application of a new analytical method, molecularly imprinted nanoparticles (nanoMIPs), to detect and determine concentrations of certain prohibited drugs, such as β-blockers, in water and human urine samples. These medications are used in the treatment of cardiovascular conditions, negative effects of adrenaline (helping to relief stress), and hypertension (slowing down the pulse and softening the arteries). They can also significantly increase muscle relaxation and improve heart efficiency. The new method of the detection and quantification of β-blockers is based on synthesis, characterization, and implementation of nanoMIPs (so-called plastic antibodies). It offers numerous advantages over the traditional methods, including high binding capacity, affinity, and selectivity for target molecules. Additionally, the whole process is less complicated, cheaper, and better controlled. The size and shape of the nanoMIPs is evaluated by dynamic light scattering (DLS) and transmission electron microscope (TEM). The affinity and selectivity of the nanoparticles are investigated by competitive pseudo enzyme-linked immunosorbent assay (pseudo-ELISA) similar to common immunoassays employing natural antibodies. To provide reliable results towards either doping detection or therapeutic monitoring using the minimal invasive method, the qualitative and quantitative analysis of these drugs is performed in water and human urine samples. It is demonstrated that the assay can detect β-blockers in water within the linear range 1 nmol·L−1–1 mmol·L−1 for atenolol with the detection limit 50.6 ng mL−1, and the linear range 1 mmol·L−1–10 mmol·L−1 for labetalol with the detection limit of 90.5 ng·mL−1. In human urine samples, the linear range is recorded in the concentration range 0.1 mmol·L−1–10 nmol·L−1 for atenolol and 1 mmol·L−1–10 nmol·L−1 for labetalol with a detection limit of 61.0 ng·mL−1 for atenolol and 99.4 ng·mL−1 for labetalol. [ABSTRACT FROM AUTHOR]