Soymilk stands out as a nutritional product and source of bioactive compounds such as isoflavones. Isoflavones, especially aglycones, are associated with beneficial effects to human health and are more easily digestible by human treatment. Therefore, there is a growing consumer interest in obtaining soy products with high isoflavone aglycone content. However, the content and isoflavone profile of soybeans and their derivatives can be influenced by certain types of processing, such as mixed culture fermentation. Therefore, the aim of this study was to evaluate the isoflavone profile in soymilk after kefir fermentation. The soymilk was fermented with mixed kefir (0.025 UC L-1) at 25°C for 15 h, control soybean extract without fermentation was also elaborated. The different forms of isoflavones were quantified in duplicate by ultra-high performance liquid chromatography (CLUE). The content of the group of β-glucosidic isoflavones (daidzin, genistin and glicitin) was higher (p <0.05) in the unfermented soymilk, with a content of 3.83 ± 0.01 μgmol.g-1 of the sample. While, the malonyl glucosides and acetyl glucosides did not present significant difference between the fermented and unfermented extract. The isoflavone content of the aglycones group (daidzein, genistein and gytetin) was higher (p <0.05) in kefir-fermented soymilk with 2.53 ± 0.01 μgmol.g-1 of the sample. This group of aglycones presented a 6-fold higher content than kefir-fermented soymilk compared to unfermented soybean extract. Thus, it was observed that the fermentation by kefir promoted the increase in the content of the group of isoflavones aglycones adding value to the extract of fermented soybean.