Assessment of pain responses and inflammation during animal surgery is difficult because traditional methods, such as visual analogue scores, are not applicable while under anaesthesia. Acute phase proteins (APPs), such as C-reactive protein and haptoglobin, that are typically monitored in veterinary research, do not show a significant change until at least 2 h post-surgery and therefore, immediate pathophysiological changes are uncertain. The current study used sequential window acquisition of all theoretical mass spectra (SWATH-MS) to investigate plasma proteome changes that occur immediately following surgery in dogs and also to assess the efficacy of a novel transdermal ketoprofen (TK) formulation. Castration was chosen as surgical model in this study. The procedure was performed on twelve dogs (n = 6 in two groups) and blood samples were collected at 0 h, 1 and 2 h after surgery for proteomic analysis. Following surgery, there was a general downregulation of proteins, including complement C- 3, complement factor B, complement factor D, transthyretin, and proteins associated with lipid, cholesterol, and glucose metabolisms, reflecting the systemic response to surgical trauma. Many of these changes were diminished in the transdermal group (TD) since ketoprofen, a non-steroidal anti-inflammatory drug (NSAID), inhibits prostanoids and the associated chemotactic neutrophil migration to site of tissue injury. SIGNIFICANCE: SWATH-MS Proteomic analysis revealed significant changes in plasma proteins, predominantly involved in early acute phase and inflammatory response at 1 & 2 h after surgery in castrated dogs. Pre-operative application of transdermal ketoprofen formulation had reduced systemic immune response, which was confirmed by negligible alteration of proteins in transdermal treated group. A key outcome of this experiment was studying the efficacy of a novel transdermal NSAID formulation in dogs.