In this chapter, we aim to develop, from a practical point of view, the interest of sedimentation field-flow fractionation (SdFFF), a subtechnique of the field-flow fractionation (FFF) family, exploiting external sedimentation fields to achieve label-free, biophysical cell sorting. Depending on the nature of the field, gravitational FFF (GrFFF) utilizes Earth’s gravity, while SdFFF applies a centrifugal multigravitational field. Since the pioneering work during the 1980s, SdFFF has emerged as a promising tool for biological separations. Despite an initially slow adoption, subsequent decades have seen a growing body of research applying GrFFF and SdFFF to a wide range of cell types, including red blood cells, stem cells, cancer stem cells, and various mammalian cells, in disciplines such as microbiology, virology, oncology, and neurology. Recently, for adherent cells the nonequilibrium gravity-assisted dynamic fractionation (Earth gravity-assisted dynamic fractionation, NEEGA-DF) was also developed. From early applications to cortical neurons to more recent advances involving induced pluripotent stem cells and mesenchymal stem cells, these techniques have demonstrated high performance in isolating rare or specific cell subpopulations from complex biological matrices. The unique advantage of FFF methods lies in their ability to separate cells based on intrinsic physical characteristics—such as size, density, rigidity, and shape—without the need for markers or labels and with total maintenance of native cell properties including cell morphology and viability. These features make them valuable tools for cell biology, diagnostics, and therapeutic applications. While fluorescence- and magnetic-assisted sorting remain gold standards, sedimentation-based FFF techniques constitute powerful alternatives, offering sterile, functional, and reusable cell populations for biotechnological and clinical uses. Thus, GrFFF and SdFFF may be considered essential, though underutilized, methodologies in the expanding field of bioseparation sciences.