It is now widely accepted that chromatin is a highly dynamic structure that participates in all DNA-related functions, including transcription, DNA replication, repair, and programmed cell death. Chromatin compaction influences plant regeneration and development. In plants, the most popular method of detecting chromatin compaction is immunofluorescence coupled with confocal microscopy. This method has great utility but still has limitations because the chromatin structure is subjected to change during nuclei isolation and treatment. To obtain in vivo status of the chromatin compaction of living cells, we explored a new method for detecting chromatin structure using flow cytometry to measure the chromatin compaction following quick protoplast isolation. This method can reflect the real condition of chromatin compaction in vivo. Using this method, we successfully and reproducibly separated various types of intact chromatin from soybean leaves at different developmental stages. The entire process is comprised of only three key steps: protoplast generation, Acridine Orange staining and flow cytometry. This approach can be completed within 6–8 h from protoplast isolation to flow cytometry measurement and shall be instrumental to plant epigenetic study.