Within the last decades conventional flow cytometry (FC) has been evolved as powerful measurement method in biology and healthcare. The imaging flow cytometry (IFC) extends the power of traditional flow cytometry by adding high resolution morphological information’s. For high efficient IFC, a stable 3D particle focusing is needed while at the same time the sample throughput is always an issue. Therefore advanced 3D focusing methods are needed in combination with powerful imaging technologies which can meet the high requirements of an IFC-System. This review gives an overview about the challenges in 3D particle focusing for IFC applications and reports about the current state in 3D particle focusing and tomographic imaging for IFC.