The lipid droplet (LD) is a cell organelle that has been linked to human metabolic syndromes and that can be exploited for the development of biofuels. The isolation of LDs is crucial for carrying out morphological and biochemical studies of this organelle. In the past two decades, LDs have been isolated from several organisms and investigated by microscopy, proteomics and lipidomics. However, these studies need to be extended to more model organisms, as well as to more animal tissues. Thus, a standard method that can be easily applied to these new samples with the need for minimal optimization is essential. Here we provide an LD isolation protocol that is relatively simple and suitable for a wide range of tissues and organisms. On the basis of previous studies, this 7-h protocol can yield 15-100 μg of protein-equivalent high-quality LDs that satisfy the requirements for current LD research in most organisms.