Network analysis has an increasing role in our effort to understand the complexity of biological systems. This is because of our ability to generate large data sets, where the interaction or distance between biological components can be either measured experimentally or calculated. Here we describe the use of BioLayout Express^3D, an application that has been specifically designed for the integration, visualization and analysis of large network graphs derived from biological data. We describe the basic functionality of the program and its ability to display and cluster large graphs in two- and three-dimensional space, thereby rendering graphs in a highly interactive format. Although the program supports the import and display of various data formats, we provide a detailed protocol for one of its unique capabilities, the network analysis of gene expression data and a more general guide to the manipulation of graphs generated from various other data types.