Many proteins act in multiple pathways which complicates phenotypic analysis. Xenopus egg extracts reconstitute complex reactions in vitro, and this can be used to develop assays that isolate a single function of a multifunctional protein. We have applied this system to study regulators of cytoplasmic dynein (dynein), which has numerous roles in the cell including trafficking, nuclear migration, and mitotic spindle formation. Here we describe a functional assay to specifically study the regulation of spindle pole self-organization by dynein and summarize an experimental approach that was used to perform a structure-function analysis of its regulator Ndel1. The approaches presented here can be generalized to isolate a single function of other multifunctional proteins.