This review covers recent developments in the preparation, manipulation, and analyses of subcellular environments. In particular, it highlights approaches for (1) separation and detection of individual organelles, (2) preparation of ultra-pure organelle fractions, and (3) utilization of novel labeling strategies. These approaches, based on innovative technologies such as microfluidics, immunoisolation, mass spectrometry and electrophoresis, suggest that subcellular analyses will soon become as commonplace as single cell and bulk cellular assays.