This protocol describes a method for nucleocytoplasmic protein tracking during normal cell cycle progression using unmanipulated, asynchronous cells. In contrast with prevalent traditional methods, our approach does not require time-consuming, perturbing cell synchronization or separation. To this end, we chose a single-cell approach and developed a flow cytometry assay that is applied to whole cells and isolated nuclei. Our protocol involves a stepwise biochemical fractionation procedure to purify nuclei from whole cells, conventional DNA and indirect immunostaining techniques for the dual labeling of cells and nuclei for DNA and protein, and a refined concept of flow cytometric data processing and calculation: through the specific combination of DNA and cell size analyses, G1, S and G2/M phases of the cell cycle are further dissected to establish a high-resolution map of cell cycle progression, to which protein expression in cells or nuclei is correlated. In a final data analysis step, cell cycle-related, cytoplasmic protein expression is calculated on the basis of results obtained for whole cells and isolated nuclei. A minimum of 8 h is required to complete the procedure. As the approach does not require cell type-restricting pretreatments, numerous cell types of different origin can be readily studied. Human amniotic fluid stem cells, primary human fibroblasts, immortalized mouse fibroblasts and transformed tumor cells are analyzed at comparable efficiencies, demonstrating low intercell assay variability.