Purpose <p>This study investigated aqueous humor (AH) levels of α-crystallins and vascular endothelial growth factor (VEGF) in patients with neovascular age-related macular degeneration (nAMD).</p> Study design <p>Prospective study.</p> Methods <p>AH samples were collected from treatment-naïve phakic nAMD patients before the first and third anti-VEGF injections and from cataract patients without retinal disease as controls. αB-crystallin, αA-crystallin, and VEGF levels were quantified using enzyme-linked immunosorbent assay. Patients were classified as good responders (achieved remission) or poor responders (persistent intraretinal or subretinal fluid) according to response after three consecutive monthly anti-VEGF injections.</p> Results <p>A total of 28 eyes from 24 nAMD patients and 27 eyes from 23 controls were analyzed. nAMD showed higher αB-crystallin and VEGF, but similar αA-crystallin levels versus controls (<i>P</i> &lt; 0.001, <i>P</i> = 0.002, and <i>P</i> = 0.721, respectively). In nAMD eyes, after two anti-VEGF injections, αB-crystallin and αA-crystallin remained unchanged, whereas VEGF decreased (<i>P</i> = 0.057, <i>P</i> = 0.182, and <i>P</i> = 0.017, respectively). Among nAMD eyes, 20 eyes from 18 patients were good responders and 8 from 6 were poor responders. Good responders demonstrated reductions in αB-crystallin and VEGF (<i>P</i> = 0.016 and <i>P</i> = 0.002, respectively), whereas poor responders showed no significant changes (<i>P</i> = 0.249 and <i>P</i> = 0.075, respectively). In untreated nAMD eyes, αB-crystallin and VEGF levels showed a positive correlation (<i>P</i> = 0.010).</p> Conclusion <p>In nAMD, AH αB-crystallin levels were elevated compared with controls. Among good responders to anti-VEGF therapy, αB-crystallin levels decreased. Moreover, αB-crystallin levels showed a positive correlation with VEGF.</p>