Heparin-induced thrombocytopenia (HIT), occurring up to approximately 1% to 5% of patients receiving the antithrombotic drug heparins, has a complex pathogenesis involving multiple partners ranging from small molecules to cells/platelets. Recently, insights into the mechanism of HIT have been achieved by using single-molecule force spectroscopy (SMFS), a methodology that allows direct measurements of interactions among HIT partners. Here, the potential of SMFS in unraveling the mechanism of the initial steps in the pathogenesis of HIT at single-molecule resolution is highlighted. The new findings ranging from the molecular binding strengths and kinetics to the determination of the boundary between risk and non-risk heparin drugs or platelet-surface and platelet-platelet interactions will be reviewed. These novel results together have contributed to elucidate the mechanisms underlying HIT and demonstrate how SMFS can be applied to develop safer drugs with a reduced risk profile.