Whole grain rye bread is a historically rich item in Latvian culture, and Latvians are rightly proud of the rye bread tradition they have cultivated. Much international research has shown that regular intake of rye bread positively influences the consumer health. Latvia has a long history of rye bread preparation, but lacks information about changes in vitamin content during the manufacturing process - from the beginning of baking till the end product - bread. The aim of the study was to investigate the changes in vitamin content during the whole grain rye bread production process. The object of the research work: traditional Latvian whole grain rye bread, the dough of which is prepared with scald and natural yeast and then baked in a wood-fired clay oven. AOAC standard methods were used to determine the content of thiamine (B1 vitamin), riboflavin (B2 vitamin), niacin (B3 vitamin), and α-tocopherol (E vitamin). The content of vitamins was analyzed in whole grain rye flour, leavened rye flour dough, and rye bread baked for 30, 45, 60 and 75 minutes. Bread crust and crumb were analyzed separately. The content of B complex vitamers in whole grain rye flour and bread was determined using LC-MS combined with stable isotope dilution assay. The study showed that whole grain rye bread contains a wide range of B complex vitamins. During the whole grain rye bread processing it was observed that, compared to vitamin content in flour, thiamine content reduced by 36%, but content of riboflavin, niacin and α-tocopherol increased by 53%, 40% and 6% respectively.