Single-molecule force spectroscopy (SMFS) technologies have become prominent as powerful tools for the investigation of the biomechanics associated with protein structure and for the study of protein unfolding pathways. In turn, the information accessed by SMFS tools provides valuable contributions to research and drug development. In particular, SMFS can aid in revealing the molecular mechanisms underlying a wide range of human pathologies that are believed to stem from the formation and aggregation of misfolded proteins, such as Alzheimer’s and Parkinson’s disease. However, current SMFS tools lack the ability to study multiple protein (un) folding events simultaneously, making the investigation of protein folding an excessively time-consuming process. Employing acoustics to manipulate and apply force on hundreds of biomolecules in parallel represents an ideal approach for obtaining high data throughput. In this application note, we present Acoustic Force Spectroscopy (AFS™) as a new technological route to perform single molecule manipulation experiments for the study of protein unfolding.