An innovative sensor technology is introduced that employs molecularly imprinted polymers (MIPs) for the electrochemical detection of ritonavir (RTV), a protease inhibitor utilized in HIV therapy. RTV is frequently used in combination with other drugs since it is also a significant inhibitor of the P450 3A4 isoenzyme. Therefore, accurate detection of RTV in complex mixtures and intricate biological matrices is necessary to evaluate the therapeutic efficacy of RTV. A polymeric layer was formed on the surface of the glassy carbon electrode (GCE) using RTV as the template molecule, methacrylic acid (MAA) as the functional monomer, and aniline in a phosphate buffer at pH 7. The morphological and electrochemical characteristics of the RTV/ANI-co-MAA@MIP-GCE sensor were assessed through scanning electron microscopy, Fourier transform infrared spectroscopy, cyclic voltammetry, and electrochemical impedance spectroscopy. The sensor exhibited a linear detection range for RTV utilizing a redox probe (5.0 mM [Fe(CN)]) spanning from 1.0 × 10 to 1.5 × 10 M, with the limit of detection and limit of quantification for standard solutions established at 2.75 × 10 M and 9.18 × 10 M, respectively. Subsequently, the sensor was effectively employed to detect RTV in commercial serum samples and tablets, yielding satisfactory recovery results. As a result, the RTV/ANI-co-MAA@MIP-GCE demonstrated high specificity, accuracy, and sensitivity in the detection of RTV. Additionally, density functional theory calculations were conducted to support the experimental results, investigating the interactions between the template and monomer, which revealed binding energies for RTV-MAA complexes at different template: monomer ratios and clarified potential intermolecular interactions.