Inference of gene regulatory networks from gene perturbation experiments is the most reliable approach for investigating interdependence between genes. Here, we describe the initial gene perturbations, expression measurements, and preparation steps, followed by network modeling using TopNet. Summarization and visualization of the estimated networks and optional genetic testing of dependencies revealed by the network model are demonstrated. While developed for gene perturbation experiments, TopNet models data in which nodes are both perturbed and measured. For complete details on the use and execution of this protocol, please refer to McMurray et al. (2021).