Cerebrospinal fluid (CSF) analysis is of significant clinical importance for the diagnosis of diseases. In humans, CSF is easily accessible and can be collected using minimally invasive methods. However, obtaining uncontaminated CSF from rats is still challenging. This study described a microsurgical technique for sampling large quantities (>200 μL) of clear and non-blood-contaminated CSF from the rat cisterna magna in a comprehensible step-by-step guide and provided a graphical visualization. CSF was sampled in 5-10 min (n = 29 animals; average surgical time 7.6 min). In visual control, 28 samples (97 %) of clear and uncontaminated CSF were obtained. The volume of CSF collected was 124-337 μL, with an average volume of 207 μL/sample. Using the Valsalva maneuver, we could collect higher volumes (up to 400 μL) several times. There is no need for special surgical skills to perform this method accurately. The method takes a few minutes longer than a percutaneous puncture (<1 min in pups). However, the volume of CSF obtained using the percutaneous approach in adult rats (50-70 μL) is comparatively low. We described a practical method of sampling CSF from rats that enables large volumes of CSF to be collected without blood contamination. No special surgical skills are required to use this method. With proper practice, the time between skin incision and CSF sampling is <10 min. Depending on the experimental design requirements, some additional time must be planned for wound closure.