In this study, molecular imprinting was used to develop a method based on noncovalent interactions for synthesis of a testosterone-specific polymer. The effect of the different template-monomer ratios, the particle sizes of polymers, and chromatographic mobile phases on steroid-polymer interactions are discussed. The polymer obtained was found to interact specifically with testosterone, while other steroids under study were eluted close to the void volume in the HPLC experiments. Batch rebinding studies in acetonitrile were undertaken to quantitatively evaluate the affinity of the polymer for testosterone. During this experiment, the testosterone concentration was measured in two ways: spectrophotometrically and by HPLC on a column with testosterone-specific imprinted polymer synthesized by us. Both methods resulted in similar values of association constants and the number of binding sites. However, the second method has obvious advantages when the analyzed solution contains a mixture of optically dense compounds. The results obtained focus on the two-point binding nature of the imprinted polymer-testosterone interaction and the significant role of hydrogen bonds between the OH group of testosterone and carboxy group of methacrylic acid residues inside specific recognition sites of the imprinted polymer.