The directional cell response to chemical gradients, referred to as chemotaxis, plays an important role in physiological and pathological processes including development, immune response and tumor cell invasion. Despite such implications, chemotaxis remains a challenging process to study under physiologically-relevant conditions in-vitro, mainly due to difficulties in generating a well characterized and sustained gradient in substrata mimicking the in-vivo environment while allowing dynamic cell imaging. Here, we describe a novel chemotaxis assay in 3D collagen gels, based on a reusable direct-viewing chamber in which a chemoattractant gradient is generated by diffusion through a porous membrane. The diffusion process has been analysed by monitoring the concentration of FITC-labelled dextran through epifluorescence microscopy and by comparing experimental data with theoretical and numerical predictions based on Fick's law. Cell migration towards chemoattractant gradients has been followed by time-lapse microscopy and quantified by cell tracking based on image analysis techniques. The results are expressed in terms of chemotactic index (I) and average cell velocity. The assay has been tested by comparing the migration of human neutrophils in isotropic conditions and in the presence of an Interleukin-8 (IL-8) gradient. In the absence of IL-8 stimulation, 80% of the cells showed a velocity ranging from 0 to 1 µm/min. However, in the presence of an IL-8 gradient, 60% of the cells showed an increase in velocity reaching values between 2 and 7 µm/min. Furthermore, after IL-8 addition, I increased from 0 to 0.25 and 0.25 to 0.5, respectively, for the two donors examined. These data indicate a pronounced directional migration of neutrophils towards the IL-8 gradient in 3D collagen matrix. The chemotaxis assay described here can be adapted to other cell types and may serve as a physiologically relevant method to study the directed locomotion of cells in a 3D environment in response to different chemoattractants.