Chloroplasts are the organelles that define plants. Along with many other metabolic, developmental and signalling functions, chloroplasts are responsible for photosynthesis, the process where sunlight energy is harnessed to power the cellular activities of life. Consequently, chloroplasts are essential, not only for plants but also for the myriad ecosystems that depend on plants, and for agriculture. Chloroplasts are composed of thousands of different proteins, most of which are nucleus-encoded and imported from the cytosol before being routed internally to clearly-distinct intraorganellar compartments. To achieve a more complete understanding of chloroplast development and functions, and to enable biotechnological strategies involving chloroplast manipulation that address global challenges linked to food security or bioenergy, it is essential to determine the targeting, localization and interactions of important chloroplast proteins. This methods collection describes a set of critically important and complementary techniques that may be used to accomplish these goals. The collection mostly focuses on the widely-used model plant Arabidopsis thaliana (Thale Cress), but the methods may also be adapted and applied to other organisms.