The versatile function of ubiquitin (Ub) is powerfully illustrated by its appearance in multiple forms and shapes, like polymeric ubiquitin chains. These chains, when recognized by specific ubiquitin-binding domains (UBDs), give rise to extraordinary complex signaling networks that regulate virtually every cellular function. At the heart of our understanding of this complexity is the evolution and adaptation of technologies and methods to analyze ubiquitin biochemistry, e.g., covalent Ub-substrate conjugates as well as transient Ub-UBD interactions. Here, we describe seminal developments in those methodologies that have paved the way to our understanding of the diversity of Ub signals as well as their recognition and interpretation by UBD-containing proteins.