Abstract: Isoflavone aglycone content increases after fermentation in soy pulp which is known for prevention of postmenopausal disease. The purpose of this study was to establish optimum fermentation conditions and suitable culture for mass production of aglycone using black soybean pulp, a by-product of soybean processing. Black soybean pulp was fermented up to 48 h using Lactobacillus acidophilus (LA) and Bacillus subtilis (BS) culture. Samples were collected every 12 h to measure isoflavones (daidzin and genistin and daidzein and genistein) using High-performance Liquid Chromatography (HPLC) and β-glucosidase activity using spectrophotometry. Isoflavone aglycone content was verified by Liquid Chromatography-tandem Mass Spectrometry (LC-MS/MS) analysis. Genistein concentrations in black soybean pulp after 12 and 24 h of fermentation with L. acidophilus were 6.8 and 7.2 fold, respectively, compared to controls. Genistein concentrations in black soybean pulp after 12 and 24 h of fermentation with B. subtilis were 6.8 and 6.9 fold greater than controls. β-glucosidase activity was highest in the black soybean pulp fermented for 12 h with L. acidophilus. Black soybean pulp fermented for 12 h with L. acidophilus was most efficient to produce isoflavone aglycone.