Red blood cells (RBCs) in sickle cell disease (SCD) vary greatly in morphological, electrical, and biomechanical characteristics. The heterogeneity in these biophysical properties can be further influenced by therapeutic interventions. Research into these properties at a single-cell level may not only enhance the understanding of clinical symptom variability and therapeutic responses but also aid in the development of novel diagnostic tools. This chapter examines the biophysical properties of single RBCs that are affected by SCD, methods for single-cell assays, and applications in SCD diagnosis. The first section introduces how hemoglobin mutation affects sickle cell membrane and interior, oxygen dependences in kinetics of cell sickling and methods to induce cell sickling in vitro. The second section reviews the differences in electrical properties between sickle cells and normal RBCs using various electrical methods, with a focus on testing principles and data analysis for measuring single sickle cells under controlled oxygen conditions. The biomechanics section covers cell deformability, viscoelastic behavior, and microfluidic models for measuring sickle cell properties. Practical applications and insights from single-cell biophysical assays for diagnosis and treatment monitoring are examined. Finally, challenges and future directions are explored, addressing the limitations of current methodologies and proposing improvements to stimulate clinical interests and research in single-cell biophysical properties as potential biomarkers of SCD and biophysical assay techniques.