Pharmacogenetics plays a critical role in personalizing medicine, particularly in optimizing drug efficacy and minimizing adverse effects. The SLCO1B1 gene, encoding the SLCO1B1 transporter, is important for the hepatic uptake of statins, influencing their pharmacokinetics and pharmacodynamics. This study investigates the frequency of SLCO1B1 c.388A>G and SLCO1B1 c.521T>C polymorphisms in the Croatian population. A total of 459 individuals of Croatian descent were genotyped using the TaqMan® method. The results revealed that the c.388A>G polymorphism is nearly evenly distributed, with allele frequencies of 52.96% for the wild-type (A) and 47.04% for the variant (G). In contrast, the c.521T>C polymorphism was less common, with a wild-type allele (T) frequency of 80.94% and a variant allele (C) frequency of 19.06%. These findings align with previous studies conducted in both Croatian and European populations. Further analysis explored the relationship between these polymorphisms and demographic factors such as gender, age, and year of birth. While the c.388A>G polymorphism showed no significant associations with these variables, the c.521T>C polymorphism demonstrated a notable gender difference in its distribution in the heterozygous genotype (521T/C). Additionally, a significant correlation was observed between the year of birth and the homozygous variant genotype (521C/C), suggesting potential generational shifts in allele frequencies. The identification of such genetic variants in the Croatian population underscores the importance of integrating pharmacogenetic testing into routine clinical practice. By tailoring statin therapy to an individual's genetic profile, healthcare providers can optimize treatment efficacy while minimizing the risk of adverse drug reactions. This approach not only enhances patient care but also has broader implications for public health, potentially leading to more effective and personalized treatment strategies in Croatia and beyond. As the understanding of pharmacogenomics continues to evolve, studies like this contribute to the growing body of evidence supporting the clinical utility of genetic testing in optimizing drug therapy and advancing personalized healthcare.