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Most microfluidic devices developed to date for the analysis of live cells incorporate channels with relatively simple constant rectangular or semi-circular cross-sections, relying on complex channel network geometries rather than alteration of the shapes of the channels themselves for development of diverse functional fluidic controls, e.g., spatial gradients of bioactive ligands. In this study we describe a simple alternative method to create highly defined and predictable gradients of surface bound molecules. This method relies on the generation of a considerable variation in the spatial distribution of flow velocities within a channel with a triangular cross-section. The triangular shape can be easily implemented by using bulk wet etching and polydimethylsiloxane (PDMS) replica molding techniques. By analytical modeling and simulation, we predict that the deposition of the solute onto a channel boundary depends on the local flow rate values, yielding gradient spanning the whole width of the channel. This prediction was validated by direct visualization of the flow rate and fibronectin–rhodamine deposition in a fabricated microchannel. Using this experimental platform, we assessed cell migration in response to a fibronectin gradient deposited in the microchannels. We find that this gradient could induce robust haptotaxis of Chinese Hamster Ovary (CHO) cells towards the areas of higher fibronectin surface density. We propose that the described simple gradient generation method can help to avoid complexity present in many current device designs, allowing to introduce more easily other potentially useful design features. [ABSTRACT FROM AUTHOR]