The blood‐brain barrier (BBB) limits substance transport to the brain and is therefore the major hurdle to overcome when developing neuroactive drugs. We report herein cerebral open flow microperfusion (cOFM) as a new membrane‐free technique for measuring substance transport across the intact BBB. cOFM is based on a probe that is inserted into the brain causing rupture of the BBB. The BBB is re‐established within 15 days and allows sampling of interstitial brain fluid under physiological conditions. The aim of this proof‐of‐concept study was (i) to determine the time between cOFM probe insertion and BBB re‐establishment; and (ii) to demonstrate the ability of cOFM to sample in the interstitial cerebral fluid with intact BBB. Re‐establishment of the BBB was determined by using Evans Blue (EB). EB is an established marker for BBB intactness as it does not cross the intact BBB. EB levels in the brain tissue indicated that the BBB was healed 11 days after probe insertion. To demonstrate transport across the healed BBB, sodium fluorescein (Naf) was used. Naf is a sensitive, low‐molecular‐weight marker that can cross the intact BBB and can be used to monitor changes in BBB permeability. Significantly increased Naf levels were found in the interstitial fluid when hyperosmolar mannitol (known to open the BBB) was introduced via cOFM, which indicated a partial opening of the BBB surrounding the cOFM probe. We show that cOFM allows monitoring of BBB permeability, which should be useful for measuring pharmacokinetics across the BBB and pharmacodynamics in the brain. This article is protected by copyright. All rights reserved.