Forster resonance energy transfer (FRET)-based biosensors have been recently applied to the study of biological pathways. In this study, a new biosensor was validated for the first time in live HEK293 and steroidogenic MLTC-1 cell lines for studying the effect of the PDE5 inhibitor on the hCG/LH-induced steroidogenic pathway. The sensor improves FRET between a donor (D), the fluorescein-like diarsenical probe that can covalently bind a tetracysteine motif fused to the PDE5 catalytic domain, and an acceptor (A), the rhodamine probe conjugated to the pseudosubstrate cGMPS. Affinity constant ( Kd) values of 5.6 ± 3.2 and 13.7 ± 0.8 μM were obtained with HEK293 and MLTC-1 cells, respectively. The detection was based on the competitive displacement of the cGMPS-rhodamine conjugate by sildenafil; the Ki values were 3.6 ± 0.3 nM (IC50 = 2.3 nM) in HEK293 cells and 10 ± 1.0 nM (IC50 = 3.9 nM) in MLTC-1 cells. The monitoring of both cAMP and cGMP by bioluminescence resonance energy transfer allowed the exploitation of the effects of PDE5i on steroidogenesis, indicating that sildenafil enhanced the gonadotropin-induced progesterone-to-testosterone conversion in a cAMP-independent manner.