Inhibitors of phosphodiesterase 5 (PDE5) are widely used for the treatment of erectile dysfunction and pulmonary hypertension. The commercially available inhibitors are effective, well-tolerated drugs, but differ in their phosphodiesterase specificity. To explore and manipulate the specificity of PDE5 inhibitors, a small library of four inhibitors was synthesized using the structure of known PDE5 inhibitors as a scaffold. Their inhibitory potency towards PDE5 and related family members was evaluated. Next, they were immobilized on a matrix to perform affinity pull-down assays in rat testis tissue, followed by mass spectrometric (MS) analysis. By using unique peptide spectral counts of identified proteins in the MS analysis, we were able to assess the relative binding of these inhibitors to a large set of proteins, allowing the determination of their selectivity profiles in vitro. For selected proteins of interest, the results were verified using quantitative isotopic dimethyl labeling and immunoblotting, and isothermal titration calorimetry (ITC). For the PDE5 inhibitors, our data reveal that even slight chemical modifications can bias their selectivity significantly towards other interacting proteins, opening up the potential of these compounds to be used as scaffolds for the development of inhibitors for new protein targets. In a broad sense, we demonstrate that the combination of chemical proteomics and unique peptide spectral counting allows for the confident and facile analysis of the differential interactome of bioactive small molecules.