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Summary: Analyzing host-microbe interactions is essential for understanding how microbiota changes disrupt host homeostasis. Here, we present a protocol for predicting host-microbe protein-protein interactions and their downstream effects using MicrobioLink. We describe steps for setting up the environment, installing software, and preparing human transcriptomic and bacterial proteomic data. The protocol outlines procedures for predicting protein-protein interactions through domain-motif interactions, integrating multi-omic datasets to map downstream effects, performing network analyses to identify key regulatory pathways, and visualizing multi-layered networks for systems-level data interpretation.For complete details on the use and execution of this protocol, please refer to Gul et al.1 and Poletti et al.2 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.