The centrosome plays a crucial role in facilitating mitotic spindle assembly through its microtubule organizing capacities. Analyzing the composition, structure, and functions of mitotic centrosomes is essential for understanding the mechanisms underlying cell division and centrosome-associated diseases. Isolating centrosomes is an effective method to gain comprehensive information about them while minimizing interference from other cellular components. In this chapter, we describe a protocol for isolating mitotic centrosomes from cultured human cells. This protocol includes cell synchronization and centrosome isolation through ultracentrifugation with a sucrose gradient. We also describe a method for conducting a microtubule nucleation assay to assess the functionality of isolated centrosomes.