Thiamine and folate are vital for both human and plant health, serving as cofactors in various metabolic processes. Thiamine is essential for carbohydrate and amino acid metabolism, while folate is crucial for DNA synthesis and the methyl cycle. Germination has been proven to increase thiamine and folate levels in lentils. While high-performance liquid chromatography (HPLC) is effective for measuring vitamin content, it does not reveal the spatial distribution of these compounds within seeds during germination. To address this, immunohistochemistry (IHC) was used in this study to localize and quantify thiamine and folate in dry, soaked, and germinated lentils. The analysis revealed that these vitamins are distributed unevenly across cotyledons and cells. In dry seeds, thiamine and folate were concentrated around starch granules, but in soaked and germinated seeds, they were dispersed throughout the cytoplasm and near protein bodies. This innovative method provided both quantitative and qualitative insights into how germination affects vitamin distribution, enhancing our understanding of seed metabolism and the behavior of vitamins during food processing.