Network analysis is a powerful tool for investigating complex interactions between different microbial taxa within a community. We present a protocol to study the gut microbial community in a mouse model of breast cancer using a network-based approach. Here, we describe the procedures for tumor cell production and inoculation and 16S rRNA data processing. We then detail steps for constructing co-occurrence networks based on correlations between microbial abundances. For complete details on the use and execution of this protocol, please refer to Wu-Chuang et al.1.