Cerebrospinal fluid flows through a channel (called the subarachnoid space) between the middle and inner layers of tissue (meninges) that cover the brain and spinal cord. To remove a sample of this fluid, a doctor inserts a small, hollow needle between two bones (vertebrae) in the lower spine, usually the 3rd and 4th or the 4th and 5th lumbar vertebrae, below the point where the spinal cord ends, and then into the subarachnoid space—the space between the layers of tissue (meninges) that cover the spinal cord (and brain). Usually, people lie on their side with their knees curled to their chest. This position widens the space between the vertebrae, so that the doctor can avoid hitting the bones when the needle is inserted.

Cerebrospinal fluid is then allowed to drip into test tubes, and the samples are sent to a laboratory for examination.