As monocytes migrate into tissues, they differentiate into macrophages. Upon activation, macrophages can polarize and adopt two very different and biologically opposing phenotypes, termed M1 and M2. M1 macrophages are considered classically activated macrophages that mediate proinflammatory responses while M2 macrophages mediate anti-inflammatory responses, both by cytokine secretion. The balance of M1 and/or M2 macrophage polarization is known to be a mediator of physiological responses in tumor progression and as such, the ability to isolate cells in the two polarization states in purified populations is crucial for understanding the dynamics of tumor progression. We show here that VisionSort can be used to isolate pure populations of M1 and M2 macrophages label-free, yielding “untouched” cells ready for downstream R&D applications.