Protoplasts have been extensively used to study replication of plant viruses at the cellular level. However, their use in studying viroid replication has been very limited because of various technical barriers. As a result, mechanisms of viroid replication remain poorly understood. The electroporation protocol presented in this unit offers a simple, fast and reproducible method to inoculate protoplasts derived from cultured cells of tobacco (Nicotiana tabacum) and the related species Nicotiana benthamiana to study viroid replication. The protocol has proven to be useful in characterizing sequence/structural features of potato spindle tuber viroid (PSTVd) that are important for replication at the cellular level, and may be adapted to study the replication of other viroids.