Abstract Axial force-spectroscopy with optical tweezers provides a natural geometry for performing rupture force measurements of protein and nucleic acid binding interactions. Axial traps, however, are typically weaker than their lateral counterparts requiring a high laser power to maintain a well calibrated, linear restoring force. Moreover, at higher forces, unless the surface tethering is carefully designed, various linkages involved in the tethering may rupture before the actual system of interest. Here we show how rupture force measurements on surface tethered molecules, up to a maximum force of ∼ 45 pN, may be designed and performed with axial optical tweezers. As both a proof of principle and a study in designing a protein handle, we explore the rupture forces involved in detaching ssDNA oligomers of varying lengths from a tethered DNA substrate. We find that the presence of short stretches of ssDNA, between the bound oligomer and substrate DNA, can significantly stabilize the binding interaction under an applied force.