The fish disease model facilitates our understanding of disease dynamics, risk assessment for disease outbreaks, the response of the gut immune system, and the maintenance of ecosystem health. Here, we present a protocol for studying gut immunity modulation by infecting Lepidocephalichthys guntea, a loach fish, with Aeromonas hydrophila. We describe steps for performing intra-peritoneal injection on fish and a bath challenge. We detail procedures for conducting periodic population calculations during the infection phase to corroborate Aeromonas hydrophila invasion in the gut. For complete details on the use and execution of this protocol, please refer to Basak and Chakraborty. 1 .