We describe a microfluidic device with rapid stimulus and lysis of mammalian cells for resolving fast transient responses in cell signaling networks. The device uses segmented gas-liquid flow to enhance mixing and has integrated thermoelectric heaters and coolers to control the temperature during cell stimulus and lysis. Potential negative effects of segmented flow on cell responses are investigated in three different cell types, with no morphological changes and no activation of the cell stresssensitive mitogen activated protein kinases observed. Jurkat E6-1 cells are stimulated in the device using r-CD3, and the resulting activations of ERK and JNK are presented for different time points. Stimulation of cells performed on chip results in pathway activation identical to that of conventionally treated cells under the same conditions.