Inertial microfluidics has been widely employed as an important sample pretreatment technique for the single-cell analysis because of the distinct advantages of label-free and external field-free operation, high-throughput processing, and simple channel structure. In this chapter, the recent advances on the application of spiral inertial microfluidics as the sample pretreatment tool for single-cell analysis will be systematically summarized. Firstly, the underlying physics of the spiral inertial microfluidics will be introduced for better understanding of the working principle. Secondly, the design guidelines will be provided for the beginners or the researchers from other disciplines to efficiently design the devices of their