This chapter discusses the strategies and approaches used to develop understanding of complex signaling networks. It focuses on two layers as templates: transcriptional regulatory processes as an ultimate controller of cell function, and protein kinase pathways as a crucial upstream "information processing" governor located downstream of environmental stimuli. Earlier investigations on binary enzyme-substrates interactions have been superseded by studies of signaling networks associated with complex and diffuse processes often resulting in subtle biological consequences. In building such network maps for higher mammalian systems pertinent to medicine, protein-protein interactions and generalized enzyme-substrate databases have formed one foundation, and transcriptional regulatory networks have formed the second foundation. The challenges in making use of these datasets lie in uncovering hierarchy of effect, integration of heterogeneous data, and the incorporation of dynamical, time-dependent data to improve the predictive value of the models. A primary control point in the regulation of differential gene expression is transcription. The basis of mapping transcriptional regulatory networks began with the development of expression arrays. Transcription is controlled by a direct and specific interaction between a transcription factor and many discrete locations within the genome, guided by a DNA sequence recognition motif. Phosphorylation of proteins by protein kinases is regulated at several levels. First, the kinase and potential ligand must be colocalized in the cell. Second, the kinase activity is generally increased when the kinase is in an active structural conformation. Phosphoproteins are of tremendous importance in eukaryotic cellular signaling and regulation. © 2010 Elsevier Inc. All rights reserved.