The lateral force separation of long chain biomolecules is demonstrated using an atomic force microscope (AFM). This is achieved by using an AFM tip to pull molecules away from the edge of a nanofluidic solution. By monitoring the torsion on the AFM cantilever, a characteristic force–distance signal is produced when long chain molecules separate and detach from the solvent edge. This lateral force separation using AFM (LFS-AFM) is demonstrated on egg albumin proteins and synthetic DNA strands. The detected length of the protein and nucleotide biopolymers was consistent with their calculated molecular contour length. LFS AFM provides separation and detection of single polymer strands that has potential applications in biochemical analysis, paleontology, and life detection.