Protamines replace histones as the main nuclear protein in sperm cells having a crucial role in compacting the paternal genome. Human spermatozoa contain the protamine 1 (P1) and the family of protamine 2 (P2) proteins. Alterations in protamine PTMs or on the P1/P2 ratio could be associated with male infertility. Top-down proteomics enables large-scale analysis of intact proteforms derived from alternative splicing, missense or nonsense genetic variants or PTMs. In contrast to current gold standard techniques, top-down allows a more in-depth analysis of protamine PTMs and proteoforms, opening up new perspectives to unravel their impact on male fertility. We analyzed two normozoospermic semen samples by top-down and discussed in detail the difficulties we found in the data analysis and the suggested solutions, as this step is one of the current bottlenecks in top-down proteomics with currently available bioinformatic tools. Our strategy for the data analysis combines two different software, ProSight PD (PS) and TopPIC suite (TP), with a clustering algorithm to decipher protamine proteoforms. We identified up to 32 protamine proteoforms at different levels of characterization. This in-depth analysis of the protamine proteoform landscape of an individual boosts personalized diagnosis of male infertility.