Mechanical properties of cells can serve as a label-free marker of cell state and function and their alterations have been implicated in processes such as cancer metastasis, leukocyte activation, or stem cell differentiation. Over recent years, new techniques for single-cell mechanical characterization at high throughput have been developed to accelerate discovery in the field of mechanical phenotyping. One such technique is real-time deformability cytometry (RT-DC), a robust technology based on microfluidics that performs continuous mechanical characterization of cells in a contactless manner at rates of up to 1000 cells per second. This tremendous throughput allows for comparison of large sample numbers and precise characterization of heterogeneous cell populations. Additionally, parameters acquired in RT-DC measurements can be used to determine the apparent Young's modulus of individual cells. In this chapter, we present practical aspects important for the implementation of the RT-DC methodology, including a description of the setup, operation principles, and experimental protocols. In the latter, we describe a variety of preparation procedures for samples originating from different sources including 2D and 3D cell cultures as well as blood and tissue-derived primary cells, and discuss obstacles that may arise during their measurements. Finally, we provide insights into standard data analysis procedures and discuss the method's performance in light of other available techniques.