Peroxisome proliferator-activated receptors (PPARs) are nuclear receptor-type transcription factors with three subtypes (α, δ, and γ) that regulate cell differentiation and metabolism. Co-crystals of human PPARα-ligand-binding domain (LBD)-PPARα ligand for X-ray crystallography have been difficult to obtain. Recombinant human PPARα-LBD proteins contain intrinsic fatty acids (iFAs of origin) and may be unstable without ligands during crystallization. To circumvent these limitations, we have successfully applied various crystallization techniques, including co-crystallization, cross-seeding, soaking, delipidation, and coactivator peptide supplementation. For complete details on the use and execution of this protocol, please refer to Kamata et al. (2020).