A comprehensive, systems-level understanding of cell signaling networks requires methods to efficiently assay multiple signaling species, at the level of single cells, responding to a variety of stimulation protocols. Here, we describe a microfluidic device that enables quantitative interrogation of signaling networks in thousands of individual cells using immunofluorescence-based readouts. The device is especially useful for measuring the signaling activity of kinases, transcription factors, and/or target genes in a high-throughput, high-content manner. We demonstrate how the device may be used to measure detailed time courses of signaling responses to one or more soluble stimuli and/or chemical inhibitors, as well as responses to a complex temporal pattern of multiple stimuli. Furthermore, we show how the throughput and resolution of the device may be exploited in investigating the differences, if any, of signaling at the level of a single-cell versus at the level of the population. In particular, we show that NF-kappaB activity dynamics in individual cells are not asynchronous and instead resemble the dynamics of the population average, in contrast to studies of cells overexpressing p65-EGFP.