The nucleus represents a fundamental feature shared by eukaryotic cells and enclosed by a highly regulated membrane barrier, the nuclear envelope (NE). Despite numerous studies elucidating the functional significance and diversity of the NE in animals and yeast, comprehensive knowledge regarding its intricate composition and functions in plants remains limited. In this chapter, we describe subtractive proteomic approach to profile novel NE components with the model organism Arabidopsis, including detailed methods for isolation of nuclei using Percoll density gradients, extraction of nuclear membranes, isolation of endoplasmic reticular enriched microsomal membranes, determination of the proteome, and validation of the enrichment at nuclear periphery. Additionally, this approach is applicable for identification of nuclear envelope protein components from diverse tissues or other plant species.