Transfer RNA (tRNA)-derived small RNAs (tDRs) are emerging as a novel class of regulatory molecules with significant implications in gene expression and cellular processes. These tDRs are generated through precise cleavage of precursor or mature tRNAs and can function in a sequence dependent manner or structure dependent manner. Recent studies have uncovered a unique subset of tDRs that can form tetramolecular assemblies, adding a new layer of complexity to their functional repertoire. Tetramolecular tDRs exhibit remarkable stability and functional diversity, influencing processes such as translation regulation, stress response, and cellular signaling. The assembly of these tDRs into tetramers is facilitated by guanine-rich sequence motifs which promote intermolecular interactions essential for their structure and biological activity. Understanding the formation, structural dynamics, and functional roles of tetramolecular tDRs offers new insights into tDR-mediated gene regulation and the potential development of RNA-based therapeutic strategies. This article aims to discuss a set of biochemical, biophysical, and reporter assay-based techniques that can be used to characterize G-quadruplex structures formed by tDRs.