In mammals, the FOXO protein family consists of four distinct isoforms: FOXO1, FOXO3, FOXO4, and FOXO6. These isoforms are key players in a wide spectrum of physiological and pathological processes, including context-specific tumor suppression. FOXO3, in particular, has emerged as a gene associated with extraordinary human longevity. While these four FOXO isoforms share common biological functions, the mechanisms underlying their overlapping and distinct roles remain less understood. It is believed that intrinsic properties and context-dependent factors contribute to isoform-specific and nonredundant FOXO functions. One promising avenue for unraveling the commonalities and specificities of these proteins involves characterizing their expression patterns in specific cell types and their activation in response to different stimuli. To facilitate this, we have developed immunocytochemistry methods capable of detecting FOXO isoforms in a highly specific manner within various human cancer cell types and fibroblasts. Importantly, this approach enables the visualization of endogenous FOXO proteins as they translocate into the cell nucleus in response to different stimuli. In this article, we present a comprehensive guide to these procedures, offering valuable insights into the distinct roles of FOXO isoforms in cellular function.