A chloroplast, delimited by a two-membrane envelope, is a type of plastid that serves as the site of photosynthesis. The photosynthetic function is based on developing an operational extensive internal membrane network, called the thylakoids, and on enzymatic processes in the chloroplast matrix, called the stroma. Thylakoid membrane is structurally different from the chloroplast envelope, and their biogenesis depends on biosynthetic and transporting activities specific to the chloroplast envelope. This kit is designed to fractionate chloroplasts into three parts (thylakoid membrane, envelope membrane, and stroma) using a specialized filter cartridge and differential centrifugation (without ultracentrifugation). Isolated thylakoids are in native forms and can be used for functional studies and the studies of subcellular localization of proteins using Western blotting/Mass spectrometry. The buffers in the kit are detergent and EDTA-free. The protocol can be completed in about one hour.