Experiments with gradients of soluble bioactive species have significantly advanced with microfluidic developments that enable cell observation and stringent control of environmental conditions. While some methodologies rely on flow to establish gradients, others opt for flow-free conditions, which is particularly beneficial for studying non-adherent and/or shear-sensitive cells. In flow-free devices, bioactive species diffuse either through resistive microchannels in microchannel-based devices, through a porous membrane in membrane-based devices, or through a hydrogel in gel-based devices. However, despite significant advancements over traditional methods such as Boyden chambers, these technologies have not been widely disseminated in biological laboratories, arguably due to entrenched practices and the intricate skills required for conducting microfluidic assays. Here, we developed microfluidic platforms integrating barriers with Quake-type pneumatic microvalves in place of microgrooves, membranes, or gels. One set of microvalves is used to maintain flow-free conditions and another set to regulate diffusion between a central channel housing the specimen of interest and sink/source reservoirs. This configuration enables stringent control over residual flows, precise spatial-temporal regulation of gradient formation, and exceptional gradient stability, maintained over extended periods