Neurons express a variety of G protein coupled receptors (GPCRs), which profoundly condition neuronal physiology by activating intracellular signalling cascades in response to extracellular signals, including neuromodulators such as noradrenaline and dopamine. One mechanism GPCR use to tune their activity is membrane trafficking. Upon ligand binding, GPCRs are endocytosed through distinct mechanisms via interaction with different proteins (e.g clathrin, endophilin). Moreover, endocytosis is affected by membrane composition. With the aim of studying endocytosis at single event level in living cells, we developed a high spatial-temporal resolution method combining pH sensitive probes and Total Internal Reflection fluorescence microscopy, called the pulsed pH (ppH) assay (A). With this, we monitored endocytosis of β-adrenergic receptors (βARs) in primary hippocampal neurons. We observed that agonist stimulation (10 µM isoproterenol) induces β2AR, but not β1AR, membrane clusters and increases only β2AR internalization rates (B). Moreover, whilst β1AR does not prefer either clathrin or endophillin2A, β2AR preferentially internalizes through clathrin. With the same approach, we investigated the effect of poly unsaturated fatty acids (PUFAs), lipids enriched in the brain, on the endocytosis of the Dopamine receptor 2 (D2R). We show that, in HEK293 cells enriched in either ω3- or ω6-PUFAs, agonist-induced (10 µM quinpirole or dopamine) D2R endocytosis, but not that of other membrane receptors, is blunted compared to control cells. Specifically, PUFA enrichment affects recruitment of β-arrestin2 at D2R endocytic sites, sparing receptor clustering at the membrane (C). Through the ppH assay, we have uncovered novel mechanistic details of GPCR endocytosis relevant to neuromodulation.