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Cell membrane-derived nanoparticles (CNPs) are a novel class of materials and are superior to synthetic nanomaterials in certain aspects due to their biological origin. Although their medical applications have been actively explored, the fundamental structure of CNPs is rarely studied. For example, the membrane orientation of CNPs is critical for their pharmacokinetics, but the previous characterizations were mostly qualitative. Herein, we report a method to quantitatively study membrane orientation of CNPs by using a 6-FAM ssDNA probe and a BHQ1 ssDNA quencher with a complementary sequence. This method utilizes specific DNA hybridization and fluorescence resonance energy transfer between 6-FAM and BHQ1. When ssDNA probes are conjugated on cell membranes, the probe marks the outer leaflet of cell membranes. The fluorescence intensities of particle solutions before and after the addition of the ssDNA quencher can be measured to quantitatively determine the fraction of CNPs with a correct outside-out (also called right-side-out) membrane orientation. Red blood cell membrane-derived nanoparticles (RBC-NPs) were fabricated and determined to have an 84% correct orientation. The quenching of membrane-bound nitrobenzoxadiazole (NBD) was used to study the permeability of RBC-NPs. It was found that RBC-NPs have a significantly higher permeability to the NBD quencher, dithionite ions, compared to live cells and egg PC/cholesterol liposomes. The ubiquitous methods using molecular probes can elucidate some structural properties of CNPs in general, enabling direct comparisons among CNPs that are derived from different cells and convenient optimization of particle fabrication. [ABSTRACT FROM AUTHOR]