Neutrophil migration from the bloodstream to sites of infection or injury is a multistep process that requires, dependent on the tissue structures being encountered, different modes of movement. Neutrophil locomotion can range from mesenchymal to amoeboid movement and may include multiple shape changes, contractile squeezing through gaps, and adhesion/de-adhesion cycles. In vitro migration assays reflect only some aspects of the complex in vivo neutrophil recruitment. For two-dimensional in vitro migration chemotaxis chambers, microscopic analysis of movement towards a pipette gradient or Boyden chambers is used. To analyze three-dimensional in vitro migration neutrophils can be embedded into matrices of diverse biophysical properties or can be placed onto matrices that are layered on a wide-pore filter, enabling migration through the matrix and the filter of a transwell plate towards a gradient of chemoattractant. We utilize here a commercially available setup for migration of murine neutrophils from the top of a loose collagen type I matrix, which determines the ability of neutrophils to attach to the matrix, sense the chemoattractant, polarize, digest the matrix, and move through the matrix into the lower transwell chamber. While the mode of migration inside the matrix cannot be studied in detail, this assay permits quantitative assessment of migrated neutrophils during a defined period of time.