Time-dependent analysis of dynamic processes in single live cells is a revolutionary technique for the quantitative studies of signaling networks. Here we describe an experimental pipeline and associated protocol that incorporate microfluidic cell culture, precise stimulation of cells with signaling molecules or drugs, live-cell microscopy, computerized cell tracking, on-chip staining of key proteins and subsequent retrieval of cells for high-throughput gene expression analysis using microfluidic quantitative PCR (qPCR). Compared with traditional culture dish approaches, this pipeline enhances experimental precision and throughput by orders of magnitude and introduces much-desired new capabilities in cell and fluid handling, thus representing a major step forward in dynamic single-cell analysis. A combination of microfluidic membrane valves, automation and a streamlined protocol now enables a single researcher to generate 1 million data points on single-cell protein localization within 1 week, in various cell types and densities, under 48 predesigned experimental conditions selected from different signaling molecules or drugs, their doses, timings and combinations.