AbstractMicrofluidic designs are versatile examples of technology miniaturisation that find their applications in various cell biology research, especially to investigate the influence of environmental signals on cellular response dynamics. Multicellular systems operate in intricate cellular microenvironments where environmental signals govern well-orchestrated and robust responses, the understanding of which can be realized with integrated microfluidic systems. In this study, we present a fully automated and integrated microfluidic chip that can deliver input signals to single and isolated suspension or adherent cells in a precisely controlled manner. In respective analyses of different single cell types, we observe, in real-time, the temporal dynamics of caspase 3 activation during DMSO-induced apoptosis in single cancer cells (K562) and the translocation of STAT-1 triggered by interferon γ (IFNγ) in single fibroblasts (NIH3T3). Our investigations establish the employment of our versatile microfluidic system in probing temporal single cell signaling networks where alternations in outputs uncover signal processing mechanisms.