Background: High grade gliomas (HGG) are incapacitating and prematurely fatal diseases. To overcome the poor prognosis, novel therapies must overcome the selective and restricted permeability of the blood–brain barrier (BBB). This study critically evaluated whether in vitro human normal BBB and tumor BBB (BBTB) are suitable alternatives to “gold standard” in vivo models to determine brain permeability. Methods: A systematic review utilizing the PRISMA guidelines used English and full-text articles from the past 5 years in the PubMed, Embase, Medline and Scopus databases. Experimental studies employing human cell lines were included. Results: Of 1335 articles, the search identified 24 articles for evaluation after duplicates were removed. Eight in vitro and five in vivo models were identified with the advantages and disadvantages compared within and between models, and against patient clinical data where available. The greatest in vitro barrier integrity and stability, comparable to in vivo and clinical permeability data, were achieved in the presence of all cell types of the neurovascular unit: endothelial cells, astrocytes/glioma cells, pericytes and neurons. Conclusions: In vitro co-culture BBB models utilizing stem cell-derived or primary cells are a suitable proxy for brain permeability studies in order to reduce animal use in medical research.