This study investigates the molecular composition and formation mechanisms of drusen, extracellular deposits that accumulate below the retinal pigment epithelium and are risk factors for age-related macular degeneration (AMD). The authors developed a method to isolate microgram quantities of drusen and Bruch’s membrane for proteome analysis. Liquid chromatography tandem MS analyses of drusen preparations from 18 normal donors and five AMD donors identified 129 proteins, with immunocytochemical studies localizing approximately 16% of these proteins to drusen. Common proteins in normal donor drusen included tissue metalloproteinase inhibitor 3 (TIMP3), clusterin, vitronectin, and serum albumin, while crystallin was more frequently detected in AMD donor drusen. Oxidative protein modifications, such as crosslinked species of TIMP3 and vitronectin, and carboxyethyl pyrrole (CEP) adducts, were observed. CEP adducts, unique to the oxidation of docosahexaenoate-containing lipids, were more abundant in AMD than in normal Bruch’s membrane and were associated with drusen proteins. Carboxymethyl lysine, another oxidative modification, was also detected in drusen. These findings support the hypothesis that oxidative injury contributes to AMD pathogenesis and suggest that oxidative protein modifications play a critical role in drusen formation.This study investigates the molecular composition and formation mechanisms of drusen, extracellular deposits that accumulate below the retinal pigment epithelium and are risk factors for age-related macular degeneration (AMD). The authors developed a method to isolate microgram quantities of drusen and Bruch’s membrane for proteome analysis. Liquid chromatography tandem MS analyses of drusen preparations from 18 normal donors and five AMD donors identified 129 proteins, with immunocytochemical studies localizing approximately 16% of these proteins to drusen. Common proteins in normal donor drusen included tissue metalloproteinase inhibitor 3 (TIMP3), clusterin, vitronectin, and serum albumin, while crystallin was more frequently detected in AMD donor drusen. Oxidative protein modifications, such as crosslinked species of TIMP3 and vitronectin, and carboxyethyl pyrrole (CEP) adducts, were observed. CEP adducts, unique to the oxidation of docosahexaenoate-containing lipids, were more abundant in AMD than in normal Bruch’s membrane and were associated with drusen proteins. Carboxymethyl lysine, another oxidative modification, was also detected in drusen. These findings support the hypothesis that oxidative injury contributes to AMD pathogenesis and suggest that oxidative protein modifications play a critical role in drusen formation.