Characterization of a nuclear proteome is a prerequisite for understanding a variety of cellular functions at the molecular level. Here, we report a novel proteomic method based on nucleus selective chemical protein labeling. In our strategy, the Hoechst nuclear staining dye is conjugated with a reactive group, which is distributed in the nucleus of live cells and selectively reacts with nuclear proteins. Using this method, we successfully identified 58 nuclear proteins, containing typical nuclear proteins Histone H2A or Antigen KI-67.