Poly(N-isopropylacrylamide) based hydrogels have been proposed as cell culture supports in cell sheet engineering. Toward this goal, we characterized the poly(N-isopropylacrylamide-co-butylacrylate) copolymer thermo-sensitivity and the cell/copolymer interactions above and below the copolymer lower critical solution temperature. We did that by direct force measurements at different temperatures using an atomic force microscope with either a polystyrene or a glass microbead as probes. We used a copolymer-coated microbead to measure adhesion after a short contact time with a single fibroblast in culture. Statistical analysis of the maximum adhesion force and the mechanical work necessary to separate the probe from the cell surface confirmed the hydrophilic/hydrophobic behavior of poly(N-isopropylacrylamide-co-butylacrylate) as a function of temperature in the range 20-37°C and, consequently, a reversible increase/decrease in cell adhesion with the copolymer. As control experiments we measured interactions between uncoated microbeads with the copolymer hydrogel or cells as well as interaction of the Poly(N-isopropylacrylamide) homopolymer with cells. These results show the potential of an assay based on atomic force microscopy for an in situ and quantitative assessment of cell/substrate interactions and support the use of poly(N-isopropylacrylamide-co-butylacrylate) copolymer as an efficient culture substrate in cell sheet engineering.