Tucupi is a traditional food from the Amazon, known for its acidic flavor, which results from the spontaneous fermentation of manipueira (cassava root liquid), a byproduct of cassava processing. However, this spontaneous fermentation lacks control over microbial activity, leading to inconsistent product quality and potential microbiological risks. Additionally, there are no studies focusing on the use of induced fermentation in tucupi production. This study aims to introduce a controlled fermentation process using isolated lactic acid bacteria (LAB) as starter cultures to standardize and improve the safety and quality of tucupi. LAB were isolated from manipueira at different fermentation stages (0h, 12h, and 24h). Samples were plated on MRS agar and incubated at 37°C for 72 hours. Colonies with coccus or bacillus morphology, catalase-negative, and Gram-positive were isolated and used as starter cultures in pasteurized manipueira fermentations, with an initial concentration of 10⁷ CFU/mL, for 24 hours. The fermentations were analyzed for pH (measured using a digital pH meter), lactic acid production and sugar content (glucose, sucrose, and fructose) using UHPLC-RI. Among the 14 isolates tested, TF10 stood out, showing the highest lactic acid production (4.25±0.00 mg/g), a pH drop from 5.94±0.04 to 3.93±0.03, and a significant reduction in glucose (from 4.40±0.01 to 0.90±0.01 mg/g), sucrose (from 7.48±0.00 to 5.89±0.00 mg/g), and fructose (from 3.48±0.01 to 0.77±0.01 mg/g). The use of the TF10 starter culture demonstrated its potential to enhance acidification, sugar metabolism, and the overall optimization of tucupi fermentation. This controlled approach provides a safer, reproducible alternative to traditional methods and offers new possibilities for scaling up tucupi production with consistent quality.