Protamine1 (P1) and Protamine 2 family (P2) are the most relevant nuclear proteins in the male gamete since are necessary to compact and protect the paternal genome. Human and mouse sperm contain a complex population of protamine proteoforms, which raises the interest to decipher whether it responds to a need of protein redundancy to ensure proper chromatin compaction, or whether specific protamine proteoforms have functional implications. In this study, we apply an optimized strategy based on Top-Down Mass Spectrometry to quantify alterations in the protamine proteoform profile of normozoospermic men with unexplained infertility and either molecular (high P1/P2) or lifestyle (obesity and advanced age) alterations. Accumulation of P2 immature forms was associated to higher P1/P2 ratios, suggesting an impaired processing of P2 in these patients. Men with obesity and intracellular oxidative stress revealed alterations in P1 proteoforms with mass shift(s) of +61 Da, either phosphorylated or not, and advanced age men showed a specific loss of diphosphorylated P1. These results point to alterations on protamine modifying mechanisms, such as proteolysis and phosphorylation, rather than on protamine synthesis, as critical factors for proper sperm chromatin maturity with impact on male fertility