We quantitatively determined interfacial potentials between cell-sized particles and stimulus-responsive hydrogels using a microinterferometer. The hydrogel is based on physically interconnected ABA triblock copolymer micelles comprising an inner biocompatible PMPC block and two outer pH-responsive PDPA blocks. The out-of-plane temporal fluctuation in the position of the cell-sized particles was calculated from changes in the interference pattern measured by Reflection Interference Contrast Microscopy (RICM), thus yielding the particle-substrate interaction potential V (Δh). Measurements in pH buffers ranging from 7.0 to 7.8 resulted in a systematic reduction in height of the potential minima 〈Δh〉 and a concomitant increase in the potential curvature V″ (Δh). The experimental data were analyzed by applying the modified Ross and Pincus model for polyelectrolytes, while accounting for gravitation, lubrication and van der Waals interactions. Elastic moduli calculated from V″ (Δh) were in good agreement with those measured by Atomic Force Microscopy. The ability to fine-tune both the gel elasticity and the interfacial potential at around physiological pH makes such triblock copolymer hydrogels a promising biocompatible substrate for dynamic switching of cell-material interactions.