Microglia are important in homeostasis and widely considered to have roles in the pathogenesis of conditions such as neuropathic pain and multiple sclerosis. The need to study microglia from the adult spinal cord is essential to further understand the role of these cells in disease pathology. Primary microglia are often prepared from brain tissues obtained from embryonic or perinatal age rodents and the process can take over a week to complete. The protocol in this study provides rapid isolation of microglia from adult spinal cord, allowing immediate availability for experimentation of both ex vivo and in vitro within a few hours. A purity of 99% with little or no neuronal or astrocytic contamination can be achieved. Between 70% and 85% of these adult microglia were in a relatively non-activated state. Functionally, these microglia respond to lipopolysaccharide incubation with increases in both phospho-p38 MAPK and OX42 immunostaining, as well as release of ATP, as compared to un-stimulated microglia. This technique provides a protocol to achieve rapid and efficient extraction of high purity, quiescent and functionally active microglia from adult mouse spinal cord, allowing greater study of adult spinal microglia in physiological and pathophysiological states.