Posttranslational mechanisms play a key role in modifying the abundance and function of cellular proteins. Among these, modification by advanced glycation end products (AGEs) has been shown to accumulate during aging and age-associated diseases but specific protein targets and functional consequences remain largely unexplored. Here, we devised a proteomic strategy to identify specific sites of carboxymethyllysine (CML) modification, one of the most abundant AGEs. We identified over 1000 sites of CML modification in mouse and primary human cells treated with the glycating agent glyoxal. By using quantitative proteomics, we found that protein glycation triggers a proteotoxic response and directly affects the protein degradation machinery. We show that glyoxal induces cell cycle perturbation of primary endothelial cells and that CML modification interferes with acetylation of tubulins and microtubule dynamics. Our data demonstrate the relevance of AGE modification for cellular function and pinpoints specific protein networks that might become compromised during aging.