Abstract: The blood-brain barrier (BBB) is a vital physiological structure that maintains the microenvironmental homeostasis in the central nervous system (CNS). Imbalances in its permeability play a key role in various neurological disorders, including stroke, neurodegenerative diseases, and brain tumors. The development of precise techniques for assessing BBB permeability is therefore paramount for elucidating the mechanisms of neurological diseases, overcoming drug development challenges and achieving precise diagnosis and treatment of CNS disorders. This review systematically summarizes the latest advances in the assessment of BBB permeability. Regarding in vitro models, platforms have evolved from the traditional Transwell system to microfluidic chips incorporating fluid shear forces, and subsequently to highly biomimetic brain organoids, with continuous improvements in the ability to simulate the neurovascular unit (NVU) microenvironment. For in vivo assessment, we detail the principles and applications of imaging techniques, including dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI), positron emission tomography (PET), second near-infrared (NIR-Ⅱ) fluorescence imaging, and two-photon microscopy (TPM), highlighting their complementary strengths in macroscopic quantification, molecular targeting, and microscopic dynamic observation. The integration of multi-modal technologies and precise quantitative assessment is a prominent trend. Future investigations will focus on artificial intelligence (AI)-driven personalized permeability assessment, the development of novel intelligent probes, and the dynamic real-time monitoring of the BBB, thereby providing powerful methodological support for neurological disease research.