Background: Phosphodiesterase-5 inhibitors (PDE5i) are the first line of drugs used for the treatment of erectile dysfunction. PDE5i inhibits the activities of phosphodiesterase-5 (PDE5) found in corpus cavernosum reducing production of guanosine monophosphate (GMP) that results in vasodilation of blood vessels thereby prolonging penile erection. Various counterfeit drugs adulterated with unknown levels of PDE5i pose a danger with reported undesirable adverse effects on patients. Methods: We developed an assay to detect counterfeit drugs containing PDE5i using spectrophotometry based on a colour change of malachite green from blue to green when exposed to inorganic phosphate (Pi) measured at 630 nm. Initially, a standard graph of a known Pi concentration was established ranging from 0 to 20 µM followed by a dual biochemical reaction system of converting GMP from cGMP by PDE5, generating Pi from the first reaction’s product by calf intestinal alkaline phosphatase (CIAP), and then measuring Pi using an equation derived from the generated standard curve. Dilutions of CX1A, a counterfeit drug sample adulterated with the PDE5i sildenafil, were prepared via ultrasonication and filtration for assay testing and evaluation. Results: Sil was detected from the two separate samples comprised of low and high dilutions of CX1A quantified as 0.673% and 0.407%, respectively, based on a standard curve of pure sildenafil established from 0.1 nM to 300 µM. Conclusions: This PDE5i assay that we developed has the potential to become an alternative analytical method for detecting PDE5i showing comparable results when evaluated using HPLC.