Biophysical cellular information at single-cell sensitivity is becoming increasingly important within analytical and separation platforms that associate the cell phenotype with markers of disease, infection, and immunity. Frequency-modulated electrically driven microfluidic measurement and separation systems offer the ability to sensitively identify single cells based on biophysical information, such as their size and shape, as well as their subcellular membrane morphology and cytoplasmic organization. However, there is a lack of reliable and reproducible model particles with well-tuned subcellular electrical phenotypes that can be used as standards to benchmark the electrical physiology of unknown cell types or to benchmark dielectrophoretic separation metrics of novel device strategies. Herein, the application of red blood cells (RBCs) as multimodal standard particles with systematically modulated subcellular electrophysiology and associated fluorescence level is presented. Using glutaraldehyde fixation to vary membrane capacitance and by membrane resealing after electrolyte penetration to vary interior cytoplasmic conductivity and fluorescence in a correlated manner, each modified RBC type can be identified at single-cell sensitivity based on phenomenological impedance metrics and fitted to dielectric models to compute biophysical information. In this manner, single-cell impedance data from unknown RBC types can be mapped versus these model RBC types for facile determination of subcellular biophysical information and their dielectrophoretic separation conditions, without the need for time-consuming algorithms that often require unknown fitting parameters. Such internal standards for biophysical cytometry can advance in-line phenotypic recognition strategies.