The development of cancer immunotherapies has been ongoing for many years and has shown limited success. Novel biomarkers are needed to identify patients most likely to respond to anticancer immune-therapeutic approaches. Moreover, a systems-level approach is required for comprehensive understanding of the interconnected components, pathways, and cell types associated with an immune response. In this chapter, we describe single cell network profiling (SCNP), a novel method for assessing and measuring immune function/dysfunction at a systems level. SCNP is a multiparametric flow-cytometry-based analysis that can simultaneously measure, at the single cell level, both extracellular surface markers and changes in intracellular signaling proteins in response to extracellular modulators. Measuring changes in signaling proteins following the application of an external modulation informs on the functional capacity of the signaling network which cannot be assessed by the measurement of basal signaling alone. In addition, the simultaneous analysis of multiple pathways in multiple cell subsets can provide insight into the connectivity of both cell signaling networks and immune cell subtypes. The experimental steps associated with an SCNP assay are (1) pre-analytical sample preparation; (2) modulation for functional analysis; (3) staining with antibody cocktail; (4) data acquisition on flow cytometer; and (5) data analysis and metrics. Important considerations for each step of the assay will be discussed, and data demonstrating the utility of SCNP for immune monitoring applications will be summarized.