In the present study we used DIA MS to characterize the profile of over 4,000 proteins in immortalized human astrocytes exposed to TNF, IL-1β, and LPS, as well as in primary human astrocytes cocultured with brain endothelial cells and exposed to LPS. For both immortalized and primary astrocytes, DIA MS data were matched against a spectral library generated by merging DDA MS runs from both unfractionated and fractionated cell lysates.