In contrast to an earlier study reporting that betulinic acid is an inhibitor of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease (Mpro), we demonstrated that beutilinic acid is not a potential Mpro inhibitor via combined approaches, including the fluorescence resonance energy transfer (FRET) assay, the fluorescence polarization (FP) assay, and the protease biosensor cleavage assay. Our results suggest that the addition of detergent to the assay buffers is essential for evaluating natural products as Mpro inhibitors. It is necessary to conduct comprehensive testing of Mpro inhibition via combined approaches for antiviral development.