The attachment of one or more ubiquitin molecules onto a target substrate, ubiquitination for short, is a posttranslational modification that determines its correct further processing within a eukaryotic cell. Ubiquitination was shown to be a central player in the regulation of many cellular processes from protein degradation to protein sorting (e.g., endocytosis), DNA repair, or other non-proteolytic pathways. Because of its importance in maintaining cellular homeostasis, it is not surprising that, in the course of evolution, pathogens, including plant pathogens such as phytoplasma, have developed strategies to manipulate ubiquitination of host targets to their own benefit. Manipulation includes the translocation of so-called effector proteins that themselves ubiquitinate host targets, thereby mediating their subsequent degradation to dampen host immune responses. The identification and characterization of phytoplasmal effector proteins that ubiquitinate target substrates, as well as the detection of a target ubiquitination is crucial to understand phytoplasma-associated pathogenesis. Here, we provide an easy-to-follow protocol adapted from a method described by Furlan and Trujillo (2017) to study the in vitro E3 ligase activity potential of phytoplasma effector proteins. Additionally, this protocol allows to study the in vitro ubiquitination of putative target substrates by phytoplasma effectors.