The elimination of amyloid beta (Aβ) oligomers is a promising strategy for therapeutic drug development of Alzheimer's disease (AD). AD mouse models that develop Aβ pathology have been used to demonstrate in vivo efficacy of compounds that later failed in clinical development. Here, we analyze the concentration and size distribution of Aβ oligomers in different transgenic mouse models of AD and in human brain samples by surface-based fluorescence intensity distribution analysis (sFIDA), a highly sensitive method for detecting and quantitating protein aggregates. We demonstrate dose- and time-dependent oligomer elimination by the compound RD2 in mouse and human AD brain homogenates as sources of native Aβ oligomers. Such ex vivo target engagement analyses with mouse- and human-brain-derived oligomers have the potential to enhance the translational value from pre-clinical proof-of-concept studies to clinical trials.