The purpose of this study is to characterize the dynamic response of fluid flow in microchannels, which can show significant delay times before reaching steady flow conditions. Two main sources of these delays are numerically and experimentally investigated, the hydraulic compliance which originates from the flexibility of the system components (microchannel, tubing, syringe, etc.), and the compressibility of the liquid dead volume in the setup, also known as the “bottleneck effect”. A fluid-structure interaction model is presented for the compliance of rectangular PDMS microchannels that is used to form a numerically based relation for the compliance as a function of the pressure and geometry. This relation is successfully able to predict the dynamics of the flow inside PDMS microchannels in stop-flow experiments. The time delays associated with the bottleneck effect is also shown when using different syringe volumes, microchannel resistances, and liquid types. In these tests, the bottleneck effect has a much larger effect compared to the compliance of the PDMS microchannels. This is true even when using softer PDMS by increasing the monomer-to-curing agent mixing ratio. The characterization that is presented here allows for a simple analysis of microfluidic networks using the hydraulic-circuit approach.