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Background Substance P (SP) is a neuropeptide that functions as a neuromodulator. It is released mainly in the brain stem and in nerve endings. Aims The present study aimed to provide fundamental data that may be applied to the assessment of pain in dogs by evaluating their serum SP concentrations. Methods Two groups of dogs were designated as pain groups that included 10 dogs with medial patella luxation (MPL) and 10 dogs with fractures, respectively, and 20 healthy dogs were enrolled as the control group. Results The SP concentrations