Darunavir (DRV) is a potent antiviral drug used for treatment of infections with human immunodeficiency virus (HIV). Effective and safe treatment with DRV requires its therapeutic drug monitoring (TDM) in patient’s plasma during therapy. To support TDM of DRV, a specific antibody with high affinity is required in order to develop a sensitive immunoassay for the accurate determination of DRV in plasma. In this study, two new and different immunogens were prepared and characterized. These immunogens were the DRV conjugates with keyhole limpet hemocyanin (KLH) protein. The first immunogen (DRV-KLH) was prepared by zero-length direct linking of DRV via its aromatic amino group with the tyrosine amino acid residues of KLH by diazotization/coupling reaction. The second immunogen (G-DRV-KLH) was prepared by conjugation of the N-glutaryl derivative of DRV (G-DRV) with KLH. The 5-carbon atoms-spacing G-DRV hapten was synthesized by reaction of DRV via its aromatic amino group with glutaric anhydride. The reaction was monitored by HPLC and the chemical structure of G-DRV was confirmed by mass, 1H-NMR, and 13C-NMR spectroscopic techniques. The hapten (G-DRV) was linked to the KLH protein by water-soluble 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) coupling procedure. The pertinence of the coupling reactions of haptens to protein was confirmed, and the immunogens were characterized by ultraviolet (UV) spectrophotometry. Both DRV-KLH and G-DRV-KLH were used for the immunization of animals and the animal’s antiserum that showed the highest affinity was selected. The collected antiserum (polyclonal antibody) had very high affinity to DRV (IC50 value = 0.2 ng mL−1; defining IC50 as the DRV concentration that can inhibit antibody binding by 50% of its maximum binding) and high specificity to DRV among other drugs used in the combination therapy with DRV. Cumulative results from direct and competitive enzyme-linked immunosorbent assay (ELISA) using this polyclonal antibody proved that the immunogens were highly antigenic and elicited a specific polyclonal antibody. The produced polyclonal antibody is valuable for the development of highly sensitive and selective immunoassays for TDM of DRV.