Human spinal pathological processes have been linked to a loss of spinal subarachnoid space (SSAS) permeability, which has therefore become a target for therapy. Hence, it has become important to measure SSAS patency in rat models of these human disorders. The estimation of in vivo rat SSAS patency is described by quantifying passage of streptavidin-covered superparamagnetic beads (SPMB) in cerebrospinal fluid (CSF). Beads are injected into the cisterna magna and recovered at spinal level L2. They are then coated with biotynilated horseradish peroxidase for enzymatically based colorimetric measurement, after removal of bloody CSF to avoid interference with the colorimetric readings. The procedure was tested in intact rats and in rats 24 h after T9 laminectomy. Residual beads in SSAS were viewed by histology. Average bead recovery from intact rats was 6.4% of amount initially administered, in a mean CSF volume of 126 μL; in laminectomized rats, it was 1%, in a mean CSF volume of 39.2 μL. Unlike in vivo imaging techniques, such as myelography (used here to validate our method) and near infrared fluorescence technology for qualitative rat SSAS patency viewing, our SPMB-based method allows for an in vivo quantitative estimation of the permeability of this space. A novel method has been established to reliably determine SSAS permeability in rats. The method is reproducible and has the required sensitivity to detect an 84.4% reduction in bead recovery, as seen in laminectomized rats compared to intact animals. Copyright © 2013 Elsevier B.V. All rights reserved.