The human HAX-1 protein is a ubiquitously expressed multifunctional protein that regulates various cellular processes through interactions with numerous cellular proteins and RNA. The purification protocol presented here is designed explicitly for HAX-1 expressed in mammalian cells and yields high amounts of soluble HAX-1 protein constructs N-terminally fused to a cleavable superfolder GFP. This protocol will enable structural studies of HAX-1, which is predicted to undergo posttranslational modifications and be partially disordered in the absence of a binding partner.