Acetylsalicylic acid and omeprazole were recently formulated by the new FDA-approved drug Yosprala ® Tablets. This novel combination was prescribed to reduce the risk of myocardial infarction in patients who were at risk for developing peptic ulcer while taking acetylsalicylic acid. In the current work, two different high precision sensitive fluorescence spectroscopic methods were developed for quantitative analysis of the above drugs in pharmaceutical dosage form and spiked human plasma. Acetylsalicylic acid was quantitatively analyzed due to its unique native fluorescence nature. The fluorescence emission of acetylsalicylic acid was quantitatively determined at 404 nm after excitation at 296 nm without any interference from omeprazole. Omeprazole, which has a free terminal secondary amino group, reacted with 4-chloro-7-nitrobenzo-2-oxa-1, 3-diazole (NBD-Cl) by a nucleophilic substitution mechanism to form a highly fluorescent dark yellow fluorophore. Omeprazole was quantitatively analyzed by measuring the emission fluorescence intensity of the dark yellow fluorophore at 535 nm after excitation at 465 nm. Various parameters affecting the described methods were carefully checked and optimized. The calibration curves were found to be linear over the concentration range of 50–1600 ng/ml for acetylsalicylic and 30–2000 ng/ml for omeprazole. The proposed methods were successfully applied to the quantitative analysis of the two drugs in the pharmaceutical dosage form Yosprala ® and in spiked human plasma.