Snake venoms are complex secretions of specialized glands that contain many biologically active substances belonging to the group of enzymes or toxins. The Nose-horned viper (Vipera ammodytes ammodytes) is the most venomous snake in Europe and also with the most recorded human bites in Europe. Its habitat spreads almost all over the Balkan Peninsula, up to 2000 m above sea level. Although the protein composition of snake venom has been studied for a long time, the venomous snakes of Europe have been relatively poorly researched. Reports of gender differences in the venom protein composition are still lacking and it is not yet certain whether they exist or are due to intraspecies individual differences. Gender differences in the venom protein composition of Nose-horned viper (Vipera ammodytes ammodytes) have not been proven or investigated so far. The aim of this study was to investigate and analyze the possible presence of sexual dimorphism in the venom protein composition of Nose-horned viper (Vipera ammodytes ammodytes). The study was conducted on the venoms of eleven male and nine female Nose-horned vipers (Vipera ammodytes ammodytes). The protein concentration of the venoms was first measured, and then the protein composition was analyzed by SDS-PAGE electrophoresis and the protein bands were densitometrically analyzed. Gender differences in protein concentration in native, undiluted samples were not statistically significant, nor were obtained total number of protein bands. Significant gender difference (male > female) was observed in the expression of the ~35 kDa protein band in samples prepared without β-mercaptoethanol. The number and expression of individual protein bands obtained by SDS-PAGE electrophoresis differ regarding the preparation of the sample itself, i.e. the addition of a reducing agent. The addition of β-mercaptoethanol as a reducing agent led to a reduction in the number of protein bands compared to samples in which no β-mercaptoethanol was added. Due to the observed individual differences in protein band number and expression, it is necessary to include a larger number of animals in further research and to consider other factors that could affect the protein composition of venoms.