Transfer RNAs (tRNAs) are short non-coding RNA molecules that play a crucial role in protein synthesis by carrying amino acids to ribosomes during translation. tRNAs are highly conserved and abundant across species, with each type categorized based on its anticodon sequence. Although traditionally viewed as essential for protein synthesis, tRNAs have been found to have additional roles in cell proliferation, tumor metastasis, and neuronal homeostasis. In addition, tRNAs are cleaved by ribonucleases to produce smaller fragments. These fragments have previously been referred to as tRNA fragments (tRF RNAs) or tRNA-derived small RNAs (tsRNAs). More recently a nomenclature has been but forward for all tRNA derived RNAs referred to as tDRs. We will use tsRNA and tDR interterchangeably. The tDRs are processed at specific sites in tRNAs and can be differentially expressed in various tissues and diseases, indicating their potential as unique non-coding RNAs with specific functions. In a previous study, we identified a 3'tDR, which can regulate the translation of a target mRNA by altering its secondary structure. This chapter provides a detailed protocol to analyze the tDR-mediated translational regulation based on several molecular methods.