Neutrophils play an essential role in the protection against infection, as they are the most numerous circulating white blood cell population and the first responders to injury. Their numbers in blood are frequently measured in the clinic and used as an indicator of ongoing infections. During inflammation and sepsis, the ability of neutrophils to migrate is disrupted, which may increase the risk of infection, even when the neutrophil count is normal. However, measurements of neutrophil migration in patients are rarely performed because of the challenges of performing the migration assays in a clinical setting. Here, we describe a microfluidic assay that measures the spontaneous neutrophil migration signatures associated with sepsis. The assay uses one droplet of patient’s blood in a microfluidic device, which circumvents the need for neutrophil isolation from blood. This assay may also be useful for the study of the effect of various immune modulators on neutrophil migration behavior from healthy volunteers and patients.