The therapeutic combination of ondansetron (OND) and pantoprazole (PAN) is widely prescribed for the treatment of many gastrointestinal tract (GIT) disorders. For this reason, a new, simple, very sensitive, accurate and precise HPTLC method was proposed for the determination of OND and PAN in pure forms, normal saline infusion solution, and spiked human plasma. Merck HPTLC aluminum plates pre-coated with silica gel 60 F254 were used as the stationary phase, and the mobile phase consisted of chloroform‒methanol‒ ethyl acetate (6:2:2 v/v). Using this system resulted in compact, symmetric peaks with the best resolution between peaks. The RF values were 0.50 ± 0.12 for OND and 0.73 ± 0.09 for PAN. The densitometric scanner was set at 302 nm using a deuterium lamp as the source of radiation. The detection and quantification limits were 7.50 and 22.50 ng per spot for OND and 15.81 and 47.42 ng per spot for PAN. The proposed method was successively applied for the analysis of the studied drugs in their pharmaceutical dosage forms, normal saline solution, and spiked human plasma. It gives excellent percent of recovery. Excellent agreement between the proposed method and the reported methods was achieved when the results were matched with respect to accuracy and precision.