The nucleolus is the location of ribosomal biogenesis, and plays crucial regulatory roles in nuclear responses to stress. Here, we report a new and improved nucleolar isolation method, which is simpler and more efficient than the traditional method. The purity of nucleoli obtained by using the new protocol is comparable to that by using the classical method, as judged by electron microscopy, Western blotting and SILAC-based quantitative proteomics. Moreover, the improved efficiency of cell harvesting in the new method, biochemical events in the nucleolus could be "frozen" and captured at precisely controlled time points. Time-lapse nucleolar proteomics after serum stimulation in HeLa cell revealed for the first time that some nucleolar proteins respond to serum stimulation within a time period as short as the first 5. min of serum re-stimulation. Proteins involved in ribosomal biogenesis and in DNA damage repair are among the most dynamic proteins during the first 10. min after serum replenishment. Notably, the proliferation marker Ki-67 is also found to enter the nucleolus after serum replenishment. To our knowledge, this is the first study that demonstrates such fast responses in the nucleolus, further confirming the rapid plasticity of this organelle.