Centrosomes, the major microtubule organizing centers, facilitate mitotic spindle formation. However, recent studies have revealed that some cancer cells lack centrosomes. These findings suggest that certain types of cancer cells drive centrosome-independent mechanisms for the assembly of mitotic spindles. Therefore, analyzing the systems of acentrosomal spindle formation is beneficial for understanding the divergence of mitotic processes in cancer cells. Here, we focus on a method for generating acentrosomal cells using the PLK4 inhibitor centrinone and describe the proper conditions, spindle pole staining markers, and microscopy settings for observing acentrosomal spindle formation in human cells.