AbstractLipid phosphoinositides are master signaling molecules in eukaryotic cells and key markers of organelle identity. Because of these important roles, the kinases and phosphatases that generate phosphoinositides must be tightly regulated. Viruses can manipulate this regulation, with the Type III phosphatidylinositol 4‐kinases (PI4KA and PI4KB) being hijacked by many RNA viruses to mediate their intracellular replication through the formation of phosphatidylinositol 4‐phosphate (PI4P)‐enriched replication organelles (ROs). Different viruses have evolved unique approaches toward activating PI4K enzymes to form ROs, through both direct binding of PI4Ks and modulation of PI4K accessory proteins. This review will focus on PI4KA and PI4KB and discuss their roles in signaling, functions in membrane trafficking and manipulation by viruses. Our focus will be the molecular basis for how PI4KA and PI4KB are activated by both protein‐binding partners and post‐translational modifications, with an emphasis on understanding the different molecular mechanisms viruses have evolved to usurp PI4Ks. We will also discuss the chemical tools available to study the role of PI4Ks in viral infection.