Biochemical reactions in eukaryotic cells occur in subcellular, membrane-bound compartments called organelles. Each kind of organelle is characterized by a unique lumenal chemical composition whose stringent regulation is vital to proper organelle function. Disruption of the lumenal ionic content of organelles is inextricably linked to disease. Despite their vital roles in cellular homeostasis, there are large gaps in our knowledge of organellar chemical composition largely from a lack of suitable probes. In this Outlook, we describe how, using organelle-targeted ratiometric probes, one can quantitatively image the lumenal chemical composition and biochemical activity inside organelles. We discuss how excellent fluorescent detection chemistries applied largely to the cytosol may be expanded to study organelles by chemical imaging at subcellular resolution in live cells. DNA-based reporters are a new and versatile platform to enable such approaches because the resultant probes have precise ratiometry and accurate subcellular targeting and are able to map multiple chemicals simultaneously. Quantitatively mapping lumenal ions and biochemical activity can drive the discovery of new biology and biomedical applications.