Chemotaxis, the movement of cells guided by chemical gradients, plays an important role in many biological processes including tumour dissemination, wound healing, neuronal patterning, and embryogenesis. Immune cells perform chemotaxis to migrate from blood to the site of infection to kill the pathogen. Cancer metastasis is another example of eukaryotic chemotaxis in which the tumour cells migrate from one place to another in response to chemical cues. Many studies were established to understand the mechanisms of chemotaxis in presence of a chemical signal, how cytoskeleton rearrangement and intracellular signalling affect chemotaxis. New and advanced experimental techniques like high-resolution confocal microscopy, microfluidics, organ on a chip will advance our understanding of eukaryotic chemotaxis. The assays in microfluidics will help us to understand better the eukaryotic system and cell migration in controlled environments. The goal of this Methods Collection is three-fold: (1) to provide state-of-the-art protocols of eukaryotic cell migration (2) eukaryotic cell migration in a controlled environment by using microfluidics (3) importance of cell-cell and cell-substrate interactions during chemotaxis. Ultimately, we believe that the in-depth techniques will improve further research and modeling in this field.