Spatial Analysis of Functional Enrichment (SAFE) is a systematic quantitative approach for annotating large biological networks. SAFE detects network regions that are statistically overrepresented for functional groups or quantitative phenotypes of interest, and provides an intuitive visual representation of their relative positioning within the network. In doing so, SAFE determines which functions are represented in a network, which parts of the network they are associated with and how they are potentially related to one another. Here, I provide a detailed stepwise description of how to perform a SAFE analysis. As an example, I use SAFE to annotate the genome-scale genetic interaction similarity network from Saccharomyces cerevisiae with Gene Ontology (GO) biological process terms. In addition, I show how integrating GO with chemical genomic data in SAFE can recapitulate known modes-of-action of chemical compounds and potentially identify novel drug mechanisms.