Many biological functions, such as the cell division cycle, are intrinsically single-cell processes regulated in part by protein synthesis and degradation. Investigating such processes has motivated the development of single-cell mass spectrometry (MS) proteomics. To further advance single-cell MS proteomics, we developed a method for automated nano-ProteOmic sample Preparation (nPOP). nPOP uses piezo acoustic dispensing to isolate individual cells in 300 picoliter volumes and performs all subsequent preparation steps in small droplets on a hydrophobic slide. This allows massively parallel sample preparation, including lysing, digesting, and labeling individual cells in volumes below 20 nl. Single-cell protein analysis using nPOP classified cells by cell type and by cell cycle phase. Furthermore, the data allowed us to quantify the covariation between cell cycle protein markers and thousands of proteins. Based on this covariation, we identify cell cycle associated proteins and functions that are shared across cell types and those that differ between cell types.Competing Interest StatementThe authors have declared no competing interest.