Hydroxysafflor yellow A (HSYA), a natural product derived from Carthamus tinctorius L., has been approved for the treatment of cardiovascular and cerebrovascular diseases and emerged as an effective therapeutic drug on myocardial ischemia reperfusion (MI/R) injury via mechanisms which are poorly understood. We employed Reductive dimethyl labeling with tandem orthogonal proteolysis-activity-based protein profiling (rdTOP-ABPP) method to reveal the direct binding proteins and site in mitochondrion.