In the world of small non-coding RNAs, tRNA-derived RNAs (tDRs) have emerged in recent years as being involved in a wide range of biological functions in every domain of life. In plants, our knowledge of the roles of tDRs is still very sparse. Nevertheless, the data produced to date demonstrate their importance in regulating gene expression at the transcriptional and post-transcriptional levels, during development, or in response to biotic and abiotic stresses. Studying the functions of plant tDRs in vivo is not an easy task, and in vitro studies offer an interesting alternative. Here we describe two in vitro approaches aimed at deciphering molecular mechanisms involving plant tDRs. On the one hand, we describe how to identify tDRs capable of inhibiting protein synthesis in vitro, and on the other, we explain how to use protoplast transfection to study the localization of tDRs and determine their protein interactome.