Optical tweezers are well suited to probe the characteristics of molecular scale interactions at the interface between biology and materials. The high resolution force and position measurements can offer unique insights into the mechanism and kinetics of adhesion between short peptide sequences, aptamers, and a variety of material surfaces used in microfluidics, medicine, and semiconductor technologies. We have developed a flexible assay to characterize the binding distribution and off-rate for a collection of peptides. The peptide sequences of interest are attached to a polystyrene bead via a 3500 bp DNA spacer using a cysteine on the peptide and a primary amine on the DNA. The modularity of the assay allows any peptide sequence containing a N-terminal cysteine to be linked to the DNA and evaluated. Casein and on certain surfaces, the DNA, act as blocking molecules to reduce non-specific adhesion. Two previously elucidated peptide sequences selected for sapphire affinity were examined and exhibited dissimilar binding distributions, mean adhesion force, and rate constants. To explore the broad applicability of the assay, the adhesion of a universal binder on slide glass was inspected.