Cyclodipeptide synthases (CDPSs) utilize two aminoacyl-tRNAs as substrates to produce diverse natural products. Here, we demonstrate that CDPSs efficiently recognize aminoacylated microhelices (miHxs) that mimic the tRNA acceptor arm. Structural and enzymological analyses using unacylated, misacylated, and engineered miHxs reveal a shared RNA-binding mode for both substrates. These findings establish miHxs as versatile tools to investigate CDPS function and, more broadly, other aminoacyl-tRNA–dependent enzymes.