Proper oxygen supply with controlled gradients, mimicking physiological conditions, is an important feature for getting reliable results from in vitro cell models in medical and pharmaceutical sciences. In this paper, microfluidic chips made from thiol-ene (TE) polymers for the generation of controlled oxygen gradients were employed, and kinetics studies of TE-induced oxygen depletion over time were performed. Dissolved oxygen concentrations inside microchannels under different flow conditions were imaged and measured with a system relying on an oxygen-sensitive dye immobilized in a foil that constituted the bottom of the microchannels. These studies revealed that at least two different processes are involved in the oxygen scavenging process, and that physisorbed water on the channel surfaces and in the immediate bulk is likely playing an important role. The results also showed that flow rate can be used to tune oxygen concentration gradients along a microchannel, with a higher flow rate (2.0 µL/min) generating a shallower gradient while a lower flow rate (0.7 µL/min) generated a steeper gradient. The obtained results provide further insight into the underlying mechanisms behind the scavenging process, but much remains still poorly understood. This includes the long-term behavior of the scavenging properties, which requires flow modulation strategies to stabilize gradients for extended experimental durations. Still, we show how TE polymers provide a practically applicable venue to generate oxygen gradients for cell-based studies in, e.g., microphysiological systems.