Abstract Background The One Health approach is emerging in response to the development of bacterial resistance. To the best of our knowledge, the possibility to use this approach in a clinical context has not yet been explored. Thus, in this paper, we report the procedures to implement a prospective observational study of diagnostic pathways in human and canine patients with suspected urinary tract infection as a means to assess the feasibility and synergistic value of setting up One Health clinical research projects and interventions. Methods/design A prospective observational study will compare different diagnostic pathways (i.e., 16 possible combinations of diagnostic tools) to gold standard in human and veterinary primary care practice in Denmark. Fifty primary care practices and 100 veterinary clinics will each consecutively include 20 human patients or 8–10 dogs, respectively. Data will be collected at practice and patient level comprising (a) information about the organization of the practice and access to different diagnostic tools, (b) information about clinical history, diagnostic path and treatment during the index consultation, (c) information about severity of symptoms during the 7–10 days following inclusion, and (d) urine culture (type of microorganism and susceptibility test). The feasibility and synergistic value of conducting future research, and/or designing common interventions, will be assessed by evaluating the comparability of human primary care and veterinary primary care with respect to study implementation and study results. Discussion Results from this study will give an insight into the feasibility and synergistic value of setting-up One Health research projects in a clinical context. This is crucial if we are to embrace the One Health approach, as a legitimate strategy to implement common interventions aimed at influencing the diagnostic process in human and canine patients in order to decrease inappropriate use of antibiotics. Trial registration The study in humans has been registered in ClinicalTrials.gov NCT02249273 .