Phytochromes are plant photoreceptors that sense red and far-red light, regulating numerous developmental processes during de-etiolation and subsequent growth. A critical aspect of phytochrome signaling involves their light-dependent translocation from the cytoplasm to the nucleus, where they mediate diverse physiological responses. Upon activation, both phytochrome A (phyA) and phytochrome B (phyB) accumulate in the nucleus, where they interact with transcription factors and modulate the localization and/or function of other regulatory proteins, such as E3 ubiquitin ligases. While the nuclear functions of phytochromes are well-documented, emerging evidence suggests they may also play signaling roles in the cytoplasm. To further elucidate the distinct roles of phytochromes in the cytoplasm and nucleus and to identify their potential interaction partners in each compartment, a protocol has been developed for isolating intact nuclei and a separate cytoplasmic fraction from Arabidopsis thaliana seedlings. This method enables the identification of interaction partners through downstream analyses, such as cross-linking combined with mass spectrometry or other protein–protein interaction techniques. The protocol involves shearing Arabidopsis seedlings followed by purification of nuclei using a Percoll gradient. This approach is relatively quick and straightforward, making it suitable for etiolated or light-exposed seedlings.