Improving the environmental friendliness of laboratory procedures has become very strongly recommended. In this research, several metric tools evaluating the analytical methods, including eco-scale, analytical greenness calculator (AGREE), green analytical procedure index (GAPI), red–green–blue software (RGB) and blue applicability grade index (BAGI) have been used to assess the greenness, performance and practicality of a new useful HPLC-DAD method. The main cause of peptic ulcers and the problems that follow is Helicobacter pylori. Due to the bacteria’s high resistance, different medications are co-administered to overcome the resistance and manage the symptoms. Two antibiotics are mostly used to decrease bacterial resistance in addition to proton pump inhibitors. Pantoprazole, Levofloxacin, and Amoxicillin are co-administered in a triple therapy regimen for suppressing Helicobacter pylori. In this work, the first-time simultaneous estimation of the three medications in rat plasma has been designed and utilized to investigate pharmacokinetics using a new HPLC method with outstanding greenness and practicability. The method involved using a C18 analytical column with an eluent of acetonitrile: 0.03 M Na2HPO4 buffer (20:80, v/v) of 1.5 mL min−1 flow rate. Oxytetracycline was used as an internal standard and the drugs were determined at 230 nm. The validation protocol was adopted according to FDA instructions, and the results proved the ability of the new method to estimate the three medications in rat plasma.