Platelets are small anucleated blood cells whose primary role is hemostasis. Lately, platelets have been associated with other functions like their involvement in innate immunity, regulation of tumor growth and viral infection, gaining increasing scientific interest. In addition, molecular mechanisms that lead to platelet activation and downstream changes are still not completely understood. After activation, platelets adhere to the damaged vessel wall or activable surface, undergo cytoskeletal reorganization, and degranulation, which leads to their complete spreading. These processes are mediated by diverse signaling molecules, among others, also with small lipids called phosphoinositides (PIs). Phosphatidylinositol 3, 5-bisphosphate [PI(3,5)P2] produced by phosphatidylinositol-3-phosphate 5-kinase (PIKfyve) regulates membrane trafficking. Phosphatidylinositol 4-phosphate (PI4P) produced by phosphatidylinositol-4-kinase-III-beta (PI4KIIIβ) and other PI4Ks is a source of a key signaling molecule, PI(4,5)P2, that also mediates actin cytoskeletal dynamics. Previous transcriptomic and proteomic data show the expression of PIKfyve and PI4KIIIβ in both mouse and human platelets. In this work, we aimed to decipher if pharmacological inhibition of PIKfyve (inhibitor YM201636) or PI4KIIIβ (IN10) prevents human platelet spreading. In addition, we analyzed downstream signaling pathways in the presence of the inhibitors after activating collagen receptor (GPVI), namely phosphorylation of Syk kinase and acetylation of tubulin. Also, we evaluated the localization of PIKfyve and PI4KIIβ (another PI4K) in human platelets in resting and activated conditions by immunostaining. We conclude that PI4KIIIβ is potentially involved in platelet spreading by modulating the cytoskeleton, however, additional studies are needed to confirm this observation.