Experimental workflow for the study of tRNAs in semen of men who inject opioid(s) daily. (1) We created a biorepository of semen and blood samples donated from people who use opioids (PWID) and those who do not. (2) From the control group, we compared different methods for isolating mature sperm cells and monitored the purity of the isolated cells by Wright and H&E staining. (3) We tested different RNA isolation methods, measured the concentration of RNA by Qubit fluorometric quantification and characterized the size and abundance of the RNA fragments by TapeStation analysis. (4) After completing these standardization assays, we purified total RNA from sperm cells from our biorepository. We also isolated total RNA from both seminal-derived exosomes (‘semen extracellular vesicles’) and from seminal plasma. (5) We applied a method called ‘phospho‐RNA‐seq’ to profile global small RNAs in sperm cell samples from PWID and from non-PWID. (6) We validated the findings from phospho-RNA-seq using crystal digital PCR. We designed specific RT primers to quantify long and short tRFs by digital PCR.