Fractionation in Gram-negative bacteria is used to identify the subcellular localization of proteins, in particular the localization of exported recombinant proteins. The process of cell fractionation can be fraught with cross-contamination issues and often lacks supporting data for fraction purity. Here, we compare three periplasm extraction and two cell disruption techniques in different combinations to investigate which process gives uncontaminated compartments from Escherichia coli. From these data, a robust method named PureFrac was compiled that gives pure periplasmic fractions and a superior recovery of soluble cytoplasmic proteins. The process extracts periplasm using cold osmotic shock with magnesium, prior to sonication and ultracentrifugation to separate the cytoplasm from insoluble material. This method handles cells cultivated in various conditions and allows preparation of active proteins in their respective compartments.