The purpose of this paper is to describe the glycosylation of ambrisentan (AMB) by cultures of Cunninghamella elegans ATCC 9245. AMB is an endothelin receptor antagonist, which is used to treat pulmonary arterial hypertension. Filamentous fungi are morphologically complex and may exhibit different forms depending on species and the nature of the culture medium. Biotransformation study was conducted to investigate the ability of C. elegans to metabolize AMB. Parameters were optimized by testing on different culture media and concentrations, pH, drug concentration, static and shaking conditions. Ambrisentan's metabolite, obtained after 240 h of incubation as a result of glycosylation pathway, was separated by HPLC and determined by high-resolution mass spectrometry. The method showed it became linear over 300–1000 μg mL−1 (r = 0.998). Accuracy, precision, robustness, and stability studies agree with international guidelines. Results are consistent in accordance to the principles of green chemistry as the experimental conditions had a low environmental impact, and few solvents use.