Bull breeding soundness evaluation (BBSE) is the most common procedure used to predict bull potential fertility. However, the use of traditional methods for semen evaluation can affect its reliability. The inclusion of additional advanced test in BBSE may increase its accuracy. This study aimed to investigate the correlation between the degree of sperm protamination and BBSE main parameters of scrotal circumference (SC), progressive motility (PM), morphologically normal sperm (NS), and different categories of morphological defects. In addition, to determine the correlation between the three methods used for protamine assessment, five Brangus bulls were subjected to the BBSE. Semen samples were collected via electro-ejaculation and evaluated using traditional methods. Three different methods were used to determine the degree of sperm protamination: aniline blue (AB) staining, chromomycin A3 staining with fluorescent microscope (CMA3-FLM), and CMA3 with flow cytometry (CMA3-FCM). Sperm protamine deficiency assessed using the three methods exhibited significant differences among bulls according to their classification by BBSE, and showed significant negative correlation with semen quality parameters of NS and PM. A significant positive correlation was found between AB positivity and morphological abnormalities. The three methods used for protamine assessment also revealed significant positive correlations. Among the three tests, AB staining was the cheapest and easiest test that offers an objective assessment method for sperm protamination. Hence, it can be concluded that the assessment of protamination using AB staining test might serve as an additional valuable parameter or a replacement whenever detail sperm motility and morphology analyses in conducting BBSE to predict bull fertility are not possible.