Constrained α-helical peptides are showing potential as biological probes and therapeutic agents that target protein–protein interactions. However, the factors that determine the optimal constraint locations are still largely unknown. Using the β-integrin/talin protein interaction as a model system, we examine the effect of constraint location on helical conformation, as well as binding affinity, using circular dichroism and NMR spectroscopy. Stapling increased the overall helical content of each integrin-based peptide tested. However, NMR analysis revealed that different regions within the peptide are stabilised, depending on constraint location, and that these differences correlate with the changes observed in talin binding mode and affinity. In addition, we show that examination of the atomic structure of the parent peptide provides insight into the appropriate placement of helical constraints.