Macrophages constantly sample tissue environments through constitutive cell surface ruffling and macropinocytosis. Detection of pathogens or danger signals activates surface receptors, including Toll-like receptors (TLRs), to generate innate immune and inflammatory responses. Using fluorescence imaging of F-actin-labelled cells and customized algorithms for quantification and analysis, we can show that TLR activation upregulates surface ruffling and macropinocytic activity. We have previously shown that Rab8a regulates TLR4 signaling by recruiting the effector, PI3Kgamma. Further analysis reveals that Rab8a and PI3Kgamma modulates signaling downstream of multiple TLRs from both the cell surface and endosomes. Here multiple approaches were used to characterize the membrane domains responsible for Rab-mediated TLR signalling. Rab8a localization in live cells shows it is on membrane ruffles and in macropinosomes, it is enriched along with the signaling phospholipid PIP3 in macropinosomes and, finally, a Rab8a FRET biosensor was created and used to pinpoint the site of TLR-mediated Rab8a activation in macropinosomes. Indeed, multiple TLR ligands activate Rab8a at this same site. The end-point for Rab8a activation is revealed to enhance the PI3K/Akt/mTOR pathway, a pathway capable of modulating the production of both pro-inflammatory and regulatory cytokines to tailor the macrophage innate immune response.