Wastewater surveillance has emerged as valuable tool for public health, with notable successes in monitoring
; SARS-CoV-2 and, more recently, antimicrobial resistance (AMR) genes. Traditionally focused on human populations,
; this approach also holds significant promise for One Health surveillance. However, the extent to which
; livestock contributes to the microbial and genetic content detected in wastewater, compared to human sources,
; remains unclear. This gap in knowledge hampers our ability to normalize and accurately interpret wastewater
; surveillance data, particularly when integrating AMR gene signals. To address this, we developed and validated two duplex droplet digital PCR methods targeting fecal indicators specific to humans and key livestock species. These methods target cytochrome b of mitochondrial DNA specific to chickens and Bacteroides DNA specific either to humans, cows or pigs. Performance was evaluated against international standards to ensure reliability and harmonization across laboratories. A fecal DNA bank of 41 samples, collected from various human and animal sources, was used to retain only specific methods. Using serial dilutions of synthetic control plasmids carrying the targeted fecal indicator sequences, sensitivity was successfully assessed, with limits of detection below 10 copies. Applicability was demonstrated using wastewater samples spiked with livestock wastes. A one- year pilot monitoring was also conducted at two Belgian wastewater treatment plants, in regions with contrasting livestock populations. This study represents an essential step toward enhancing wastewater surveillance by integrating robust key species-specific fecal indicators. It lays the foundation for improved surveillance of livestock-associated outbreaks and the spread of AMR within a One Health framework.