Background: Gluten in wheat, barley, and rye can cause adverse reactions in individuals with Celiac disease, who need to follow a strict gluten-free diet. There are regulations in place that defines gluten-free. Accurate quantification of gluten in different foods, including fermented and hydrolyzed foods, is necessary to comply with the gluten-free regulations. Here we report a multiplex-competitive ELISA for the quantification of gluten in fermented sourdough. Methods: Gluten-incurred sourdough using four types of sourdough starters were prepared. The starter cultures were combined with rice flour, water, incurred with 8, 20, and 100 ppm wheat gluten, and fermented for 72 hrs. Samples collected every 24 hours were analyzed by a multiplex-competitive ELISA that uses a gluten-incurred yogurt calibrant and six gluten specific antibodies. Gluten concentrations in the samples were estimated using a four-parameter logistic (4 PL) regression. Results: The quantitative values from two out of six antibodies used in the ELISA were used to estimate the gluten concentrations in the sourdough samples. The average gluten recovery was between 55-195% for all samples. The coefficient of variation (%CV) ranged between 2-31%. Implications: The average % recovery for most sourdough samples were within acceptable range of 50-150%. Few samples showed recovery up to 200%. The %CV for most samples were ≤ 20%. These results indicate that the multiplex-competitive ELISA can provide accurate and precise quantitation of wheat gluten in fermented sourdough. The effects of heat treatment and several other variations in sourdough preparation on quantitation is currently being evaluated.