Various physiological and pathological processes are accompanied with the alteration of extracellular local pH. Accordingly, there has been a strong demands for the development of methods to analyze the cell surface pH. We established a novel method of in vitro cell surface pH imaging by using a membrane-anchored pH probe, poly(ethylene glycol)-phospholipid conjugated with fluorescein isothiocyanate (FITC-PEG-lipid). When added into the cell culture medium, FITC-PEG-lipid is spontaneously inserted into the plasma membrane via its phospholipid moiety, and retained at the extracellular surface. The ratiometric readout of its fluorescence was unique to the extracellular pH in the range of weakly alkaline and acidic pH. Our study demonstrated that FITC-PEG-lipid is useful as a sensitive and reversible cell-surface-anchored pH probe. The simple cell-surface labeling procedure of FITC-PEG-lipid is advantageous especially when considering its application to high-throughput in vitro assay. Furthermore, PEG-lipid holds a great potential as the membrane anchor of various analytical probes to approach the juxtamembrane environments.