FOXO transcription factors respond to a number of different stresses by shuttling from the cytoplasm to the nucleus where they upregulate hundreds of target genes with diverse cellular functions. The cellular consequences of FOXO activation are both stress and cell-type specific. Recent evidence suggests that one way in which FOXO dictates stress-specific outcomes is through distinct nuclear/cytoplasmic shuttling dynamics. Here we outline methods for measuring FOXO nuclear shuttling dynamics using fluorescence-based reporters.