In this work, an ultra-performance liquid chromatographic method was developed and subjected to bioanalytical method validation for screening the root extract of Ashwagandha. The method was also extended for simultaneous determination of Sildenafil, Vardenafil and Avanafil in plant root extract and in human plasma samples to detect the possible counterfeiting of the plant's pharmaceutical products with other synthetic drugs that might endanger the life of their consumers, particularly elderly people, and those with chronic diseases. The method was developed using BDS Hypersil TM C18 column (250 × 4.6 mm and 3 μm particle size) as a stationary phase and a mobile phase consisting of acidified water and acetonitrile in a gradient elution program with a flow rate of 1.00 mL min 1. In the plant extract, the proposed method was found to be linear in the range 1.00–200.0 μg mL−1 for the Vardenafil and Avanafil and 5.00–200.0 μg mL−1 for Sildenafil with a recovery percentage in range of 98.18–101.07 % for the three drugs. Intra-day and inter-day precision ranges were 0.088–1.219 and 0.151–1.313, respectively. The limit of detection and the limit of quantification for Vardenafil and Avanafil were 0.166 μg mL−1 and 0.500 μg mL−1, respectively. Whereas, for Sildenafil 0.333 μg mL−1 and 1.000 μg mL−1. The ICH validation parameters were also demonstrated. Bioanalytical method validation was established following US-FDA recommendations and the method showed good linearity in the range 1.00–200.00 μg mL −1 for Vardenafil and Avanafil, and 5.00–200.00 μg mL −1 for Sildenafil. The results revealed high accuracy with % recovery ranging from 91.05 to 98.91 % for the three drugs. The intra-day and inter-day precision were also conducted, in terms of % relative standard deviation, and ranged from 0.119 to 4.037 % and from 0.223 to 5.062 %, respectively. Furthermore, other validation parameters were evaluated including extraction recovery and stability of the analytes throughout samples preparation and storage for both short- and long-term. The method greenness was evaluated using NEMI, GAPI, AGREE and analytical eco-scale.