Keratinocytes are crucial for skin protection thus overall health. They are often modeled in cell culture studies to promote structural integrity and wound healing purposes. This study aims to develop precise methods using magnetic levitation technique for measuring keratinocyte density at the single cell level and to investigate how variations in measurements affect keratinocyte behavior and function in normal and pathological conditions. In addition, through MagLev technology, we seek to create 3D keratinocyte constructs and assess their structural integrity and functionality for tissue engineering. In this study four different HaCaT cell groups were used to examine their behavior in a magnetic field. Our single cell density measurements revealed significant differences, particularly in the G45E group, demonstrating standardization and reproducibility for HaCaT cells. Previous studies have determined the density of various cell types, but keratinocytes were examined for the first time in this context. In biofabrication experiments, significant levitation height differences were noted in the G45E group, further confirming our experimental standardization. Magnetic levitation culture transferred to agarose resulted in more consistent spheroid structures compared to direct transfer, which formed disorganized structures. Thus, keratinocyte structures maintained their integrity with magnetic levitation, facilitating further 3D culture. The results improve the understanding of the biophysical properties of keratinocytes and demonstrate that label-free, scaffold-free magnetic levitation can be a practical alternative for tissue engineering. This approach via negative magnetophoresis offers significant potential in bioengineering at the cell and tissue level to create complex 3D structures without conventional scaffolds, providing a novel, density-based detection method.