Post-translational modifications on histone proteins play critical roles in the regulation of chromatin structure and all DNA-templated processes. Accumulating evidence suggests that these covalent modifications can directly alter chromatin structure, or they can modulate activities of chromatin-modifying and -remodeling factors. Studying these modifications in the context of the nucleosome, the basic subunit of chromatin, is thus of great interest; however, the generation of specifically modified nucleosomes remains challenging. This is especially problematic for most structural biology approaches in which a large amount of material is often needed. Here we discuss the strategies currently available for generation of these substrates. We in particular focus on novel ideas and discuss challenges in the application to structural biology studies.