Changes in environmental conditions alter host–parasite interactions, raising the need for effective epidemiological surveillance. Developing operational, accurate, and cost-effective methods to assess individual infection status and potential for pathogen spread is a prerequisite to anticipate future disease outbreaks in wild populations. For endoparasites, effective detection of infections usually relies on host-lethal approaches, which are barely compatible with wildlife conservation objectives. Here, we used the brown trout (Salmo trutta)—Tetracapsuloides bryosalmonae host–parasite system to develop a non-lethal method for endoparasite infection detection, hereafter called “uDNA” for urine DNA. The uDNA diagnostic test is based on the amplification of endoparasite DNA from host urine. We sampled wild fish (N = 111) from eight sites, let them excrete in individual buckets filled with mineral water and performed parasite DNA amplification from water filtration. We compared the results of the uDNA diagnostic test for host infection status and parasite load to those from kidney samples (the current standard method). uDNA was sensitive in determining host infection status (even for infected hosts showing no sign of the disease), since up to 90% of fish individuals were correctly assigned to their infection status. The quantity of uDNA detected from the hosts depended on the sampling sites, suggesting a spatial variation in the parasite spread. uDNA was positively, but weakly correlated with parasite load in the kidney. This correlation depended on the severity of macroscopic lesions caused by the disease and was negative in fish with severely damaged kidney, likely due to impaired urine excretion. The uDNA approach provides novel avenues to non-lethally infer infection parameters from wildlife populations at large spatial scales. By targeting parasite transmission stage, uDNA is also valuable to get insights on the parasite fitness and the ecological and evolutionary dynamics of this host–parasite interaction.