Advances in both basic cell biology and point-of-care diagnostic technology led to the need for reliable, accurate, and fast techniques to separate single cells from a heterogeneous mixture. Conventional cell separation techniques, such as fluorescence-activated cell sorting (FACS) and magnetic-activated cell sorting (MACS), are very efficient methods. However, these techniques are generally expensive, require bulky equipment, and are not suitable for use in field settings. On the other hand, microfluidic technology matches the performance of existing techniques, while being more portable and suitable for field use. In this chapter, we discuss the working principles and illustrate some applications of various established and emerging microfluidic techniques. The emphasis of this chapter is more on the recent advancement in the field of microfluidics for rare or single-cell separation.