The pathogenesis of glaucoma is still unknown. There are few studies on the dynamic change of tissue-specific and time-specific molecular pathophysiology caused by ocular hypertension (OHT). This study aimed to identify the early proteomic alterations in the retina, optic nerve head (ONH), and optic nerve (ON). After establishing a rat model of OHT, we harvested the tissues from control and glaucomatous eyes, and analyzed the changes in protein expression using a multiplexed quantitative proteomics approach (TMT-MS3). Our study identified 6403 proteins after 1-day OHT and 4399 proteins after 7-day OHT in the retina, 5493 proteins after 1-day OHT and 4544 proteins after 7-day OHT in ONH, and 5455 proteins after 1-day OHT and 3835 proteins after 7-day OHT in the ON. Of these, 560 and 489 differential proteins were identified on days 1 and 7 after OHT in the retina, 428 and 761 differential proteins were identified on days 1 and 7 after OHT in the ONH, and 257 and 205 differential proteins on days 1 and 7 after OHT in the ON. Computational analysis on days 1 and 7 of OHT revealed that alpha-2 macroglobulin was upregulated across two time points and three tissues stably. The differentially expressed proteins between days 1 and 7 after OHT in the retina, ONH, and ON were associated with glutathione metabolism, mitochondrial dysfunction/oxidative phosphorylation, oxidative stress, microtubule, and crystallin. And the most significant change in retina are crystallins. We validated this proteomic result with the western blot of crystallin proteins and found that upregulated on day 1 but recovered on day 7 after OHT, which are promising as therapeutic targets. These findings provide insights into the time- and region-order mechanisms that are specifically affected in the retina, ONH, and ON in response to elevated IOP during the early stages.Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.