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The cellular response to a molecular stimulus depends on the concentration and duration of the stimulus. Conventional assays often neglect timing because the experiments are prohibitively tedious and time consuming. In this work, we describe a microfluidic living cell assay that allows efficient characterization of stimulus timing. A microfluidic network is designed to allow controlled parallel delivery of different stimulus regimens to downstream linear arrays of living GFP reporter cells. In contrast to conventional techniques that require a separate experiment for each exposure regimen and each response time point, this assay screens exposure durations and response dynamics in a single experiment. We demonstrate the approach by studying the effect of 7 durations of TNF-alpha exposure on activation of NFKB-mediated transcription at 17 time points, equivalent to 114 conventional experiments. This general approach to studying cell signaling dynamics has significant scaling potential and will be useful for high throughput drug development and investigation of dynamic signaling in basic cell biology.