Most protocols for yeast subcellular fractionation involve the use of mechanical shear forces to lyse the spheroplasts produced by the enzymatic digestion of the Saccharomyces cerevisiae cell wall. These mechanical homogenisation procedures often involve the manual use of devices such as the Douncehomogeniser and so are very operator-dependent and, in consequence, lack reproducibility. Here, we report a highly reproducible method of homogenising yeast cells based on nitrogen cavitation. This has been optimised to allow efficient release of subcellular compartments that show a high degree of integrity. The protocol remains effective and reproducible across a range of sample volumes and buffer environments. The subsequent separation method, which employs both sucrose and iodixanol density gradients, has been developed to resolve the major membrane-bound compartments of Saccharomyces cerevisiae. We present an integrated protocol that is fast, facile, robust, and efficient and that will enable 'omics studies of the sub-cellular compartments of Saccharomyces cerevisiae and other yeasts. This article is protected by copyright. All rights reserved.