The methods used for invertase activity determination are based on the measurement of glucose or reducing sugars produced by the enzymatic hydrolysis of sucrose into glucose and fructose. When whole yeast cells are used in these assays, the monosaccharides formed by the action of the periplasmic enzyme can be taken up and metabolized, leading to errors on the enzyme activity determination. This study reports a method for a more accurate invertase activity measurement by blocking the glycolytic pathway. In this method the cells were preincubated with 50 mM sodium fluoride, and inhibitor of enolase. This in vivo measurement of the enzyme activity, under initial rate conditions, was performed using cell concentrations up to 64 mg cell/ml. The results obtained showed that this method is particularly useful for cells with low invertase activity.