The objective of this study was to provide a comprehensive assessment of protamine isoforms and modifications in human sperm with the aim of identifying how protamine modifications/isoforms are altered in men with reduced sperm motility and low sperm count. Sperm samples from 18 men with prior reported pregnancy and normozoospermia 14 men from couples with infertility and asthenozoospermia and 24 men from couples with infertility and oligoasthenoteratozoospermia were included in the study. Proteomic assessment using both top-down and bottom-up LC-MS was performed.