Investigation of biological processes at the single cell or subcellular level is critical in order to better understand heterogeneous cell populations. Nanoscale secondary ion mass spectrometry (NanoSIMS) enables multiplexed, quantitative imaging of the elemental composition of a sample surface at high resolution (<50 nm). Through measurement of two different isotopic variants of any given element, NanoSIMS provides nanoscale isotope ratio measurements. When coupled with stable isotope tracer methods, the measurement of isotope ratios functionally illuminates biochemical pathways at suborganelle resolution. In this review, we describe the practical application of NanoSIMS to study biological processes in organisms ranging from microbes to humans, highlighting experimental applications that have provided insight that is largely unattainable by other methods.