ATR–ATRIP is activated upon binding to TopBP1, and this activation is driven by TopBP1-mediated autophosphorylation. To identify the phosphorylation sites associated with ATR–ATRIP activation, we prepared ATR–ATRIP samples before and after autophosphorylation and performed nano-HPLC–MS/MS analysis to map and compare the phosphorylation sites between the two samples.