The analysis of isolated organelles is one of the pillars of modern bioanalytical chemistry. This review describes recent developments on the isolation and characterization of isolated organelles both from living organisms and cell cultures. Salient reports on methods to release organelles focused on reproducibility and yield, membrane isolation, and integrated devices for organelle release. New developments on organelle fractionation after their isolation were on the topics of centrifugation, immunocapture, free flow electrophoresis, flow field-flow fractionation, fluorescence activated organelle sorting, laser capture microdissection, and dielectrophoresis. New concepts on characterization of isolated organelles included atomic force microscopy, optical tweezers combined with Raman spectroscopy, organelle sensors, flow cytometry, capillary electrophoresis, and microfluidic devices.