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One of the main objectives of root canal treatment is to alleviate the pain associated with irreversibly inflamed pulps. However, some patients may have moderate to severe pain following treatment. The aim of this study was to compare and assess the effect of ketorolac tromethamine on substance P expression in the pulp and periapical tissues when used as a root canal irrigant for single-visit root canal treatment in teeth with irreversible pulpitis. Thirty-six patients were randomly allotted to three irrigant groups – saline (n = 14), 3% sodium hypochlorite (n = 11) and ketorolac tromethamine (n = 11). Pulp blood samples (S1) were collected on gaining access to the pulp, and periapical blood samples (S2) were collected after root canal preparation. Quantification of substance P was done by ELISA test. The ketorolac tromethamine group had greater reduction in substance P expression (S2). Post-operative pain levels were not significantly influenced by the different root canal irrigants.