Understanding peptidyl-tRNA and the enzymes responsible for recycling them has come from the ability to detect and quantify enzymatic peptidyl-tRNA hydrolysis. The methods available to study removal of peptides from tRNA have evolved considerably. Radioactive [14C] amino acids were first implemented to monitor cleavage of the peptide-nucleotide ester bond of uniform peptidyl-tRNA substrates. Later, Northern blots with radiolabeled oligonucleotide probes were used to observe cleavage of specific peptidyl-tRNAs or individual tRNA from bulk peptidyl-tRNA populations. Finally, the use of fluorescently labeled amino acids was introduced, which could be coupled to anisotropy or PAGE readouts. Here we review the methods for quantification and analysis of enzymatic peptidyl-tRNA hydrolysis and summarize their inherent advantages and disadvantages.