Cerebrospinal fluid (CSF) dynamics hold implications for neurological health. Despite its importance, accurate quantification of the CSF secretion rate remains a challenge due to methodological controversies and the influence of anesthesia. A novel technique is established to determine CSF dynamics in awake and freely moving rats, and the CSF secretion is quantified with three different methodologies. The CSF secretion rate is higher in awake rats than in anesthetized rats, the latter demonstrating no requirement for mechanical ventilation for optimal CSF quantification. The CSF secretion rate is ≈10-fold lower with the "direct method" than with the ventriculo-cisternal perfusion assay, although the relative acetazolamide-mediated reduction in CSF secretion is similar across three tested methods. The findings demonstrate the importance of awake models for optimal quantification of the absolute rate of CSF secretion but highlight the versatility of methodologies for the determination of relative changes in CSF secretion associated with inhibitors, age, sex, and various pathologies.