In human rRNA, at least 104 specific uridine residues are modified to pseudouridine. Many of these pseudouridylation sites are located within functionally important ribosomal domains and can influence ribosomal functional features. Until recently, available methods failed to reliably quantify the level of modification at each specific rRNA site. Therefore, information obtained so far only partially explained the degree of regulation of pseudouridylation in different physiological and pathological conditions. In this focused review, we provide a summary of the methods that are now available for the study of rRNA pseudouridylation, discussing the perspectives that newly developed approaches are offering.