The blood-brain barrier (BBB) plays a key role in the health of the central nervous system. Opening the BBB is very important for drug delivery to brain tissues to enhance the therapeutic effect on brain diseases. It is necessary to in vivo monitor the BBB permeability for assessing drug release with high resolution; however, an effective method is lacking. In this work, we developed a new method that combined spectral imaging with an optical clearing skull window to in vivo dynamically monitor BBB opening caused by 5-aminolevulinic acid (5-ALA)-mediated photodynamic therapy (PDT), in which the Evans blue dye (EBd) acted as an indicator of the BBB permeability. Using this method, we effectively monitored the cerebrovascular EBd leakage process. Moreover, the analysis of changes in the vascular and extravascular EBd concentrations demonstrated that the PDT-induced BBB opening exhibited spatiotemporal differences in the cortex. This spectral imaging method based on the optical clearing skull window provides a low-cost and simply operated tool for in vivo monitoring BBB opening process. This has a high potential for the visualization of drug delivery to the central nervous system. Thus, it is of tremendous significance in brain disease therapy. Monitoring the changes in PDT-induced BBB permeability by evaluating the EBd concentration using an optical clearing skull window. (A) Entire brains and coronal sections following treatment of PDT with/without an optical clearing skull window after injection of EBd. (B) Typical EBd distribution maps before and after laser irradiation captured by the spectral imaging method. (Colorbar represents the EBd concentration).