Network science is a cross-disciplinary domain that focuses on the representation, analysis and modeling of complex systems as networks or graphs. In computational biology, Cytoscape is a very popular open source software platform for network modeling and visualization. As an example, Cytoscape can be used to identify disease modules which are defined as connected subnetworks showing mechanistic evidence for a specific phenotype.In the context of a new subnetwork identification, it is of interest to have access to measures of connectivity that help to assess the probability that this subnetwork could arise by chance.The R subnetsim package uses bootstrapping to test the hypothesis that a specific subnetwork is more connected than randomly generated subnetworks with the same number of nodes.Three measures of connectivity are validated by the R subnetsim package: - the number of nodes in the subnetwork including all the first-degree neighbor nodes - the number of links in the subnetwork including all the links of the first-degree neighbor nodes - the number of links in the subnetworkThe R subnetsim package also offers the possibility to easily graph the results of the simulations.This protocol provides the detailed method of a simulation analysis done on a specific subnetwork. To do so, the global subnetwork, from which the subnetwork is extracted, is needed in SIF format file.