Acid-related gastric diseases are associated with disorder of digestive tract acidification due to the acid secretion by gastric proton pump, H+,K+-ATPase. Omeprazole is one of the persuasive irreversible inhibitor of the proton pump H+,K+-ATPase. However, the reports on the mechanistic pathway of irreversible proton pump inhibitors (PPIs) on the acid activation and formation of disulfide complex are scarce in the literature. We have examined the acid activation PPIs, i.e., timoprazole, S-omeprazole and R-omeprazole using M062X/6-31++G(d,p) in aqueous phase with SMD solvation model. The proton pump inhibitor is a prodrug and activated in the acidic canaliculi of the gastric pump H+,K+-ATPase to sulfenic acid which can either form another acid activate intermediate sulfenamide or a disulfide complex with cysteine amino acid of H+,K+-ATPase. The quantum chemical calculations suggest that the transition state (TS5) for the disulfide complex formation is the rate-determining step of the multistep acid inhibition process by PPIs. The free energy barrier of TS5 is 5.5 kcal/mol higher for timoprazole compared to the S-omeprazole. The stability of the transition state for the formation of disulfide bond between S-omeprazole and cysteine amino acid of H+,K+-ATPase is governed by inter- and intramolecular hydrogen bonding. The disulfide complex for S-omeprazole is thermodynamically more stable by 4.5 kcal/mol in aqueous phase compared to disulfide complex of timoprazole, which corroborates the less efficacy of timoprazole as irreversible PPI for acid inhibition process. It has been speculated that sulfenic acid can either form sulfenamide or a stable disulfide complex with cysteine amino acid residue of H+,K+-ATPase. The M062X/6-31++G(d,p) level of theory calculated results reveal that the formation of tetra cyclic sulfenamide is unfavored by similar to 17 kcal/mol for S-omeprazole and 11.5 kcal/mol for timoprazole compared to the disulfide complex formation in each case. The DFT calculations have further shed light on the acid activation process of R- and S-isomers of omeprazole. The calculated results suggest that the efficacy of these isomers lie on their metabolic pathway and excretion from human body.