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Tartrazine (E102) is a synthetic food coloring, which belongs to the class of mono azo dyes and is known to cause numerous health problems. The current research aimed to evaluate the effect of this food dye on the enzymatic activity of amylase, lipase and proteases after a subchronic ingestion in Swiss mice. Additionally, an in vitro digestion model was used to highlight the relationship between the probable toxicity of tartrazine and the nature of the food ingested. The results show that there were no adverse effects of tartrazine on the body weight gain, and on amylase or lipase activities. However, in the high dose of tartrazine (0.05%) group, a significant decrease in trypsin and chymotrypsin enzymatic activities were observed. Regarding the in vitro digestion model, our findings show that there were no changes in the trypsin and chymotrypsin enzymatic activities either using 7.5 or 75 mg of tartrazine mixed with rice, butter or milk. We conclude that excessive consumption of tartrazine appears to alter the enzymatic activity of proteases in vivo which may have deleterious consequences on digestion. Even thought the dose close to the acceptable daily intake does not affect those activities, a strict control of tartrazine dose in high-consumption foods especially among children is an indispensable task.