Acidification of the extracellular juxtamembrane space is involved in variousbiological processes. However, imaging probes to investigate the spatio-temporaldynamics as well as the functional significance of cell surface pH are stilllimited. We established a method of cell surface pH imaging by using amembrane-anchored probe, poly(ethylene glycol)-phospholipid conjugated withfluorescein isothiocyanate (FITC-PEG-lipid). When added into the cell culturemedium, FITC-PEG-lipid was inserted into the plasma membrane via itsphospholipid moiety. FITC-PEG-lipid was retained at cell surface. Theratiometric readout of fluorescence was reversible and unique to theextracellular pH in the range of neutral and weakly acidic pH. The measurementwith FITC-PEG-lipid was accurate enough to distinguish the difference of 0.1 pHunit near the inflection point of fluorescence ratio. Our study demonstratesthat FITC-PEG-lipid is useful as a cell surface-anchored pH probe. The simplelabeling procedure is advantageous especially when considering its applicationto high-throughput in vitro assay. Furthermore, PEG-lipid holds a greatpotential as the membrane anchor of various analytical probes to investigate thejuxtamembrane environments.