The aim of this work was to develop a spectrophotometric method for the measurement of salivary β-galactosidase for the evaluation of oral malodor. A comparison between different methods for estimating oral malodor has been conducted on 94 healthy adult volunteers. For organoleptic measurements, the subjects were instructed to exhale briefly through the mouth at a distance of approximately 10 cm from the noses of two trained judges. The evaluation of β-galactosidase activity was accomplished in unstimulated whole saliva of all participants through the colorimetric method and spectrophotometry. A significant association among spectrophotometric evaluation of β-galactosidase and organoleptic measurements was assessed by Spearman correlations. Although colorimetric and spectrophotometric assessments of β-galactosidases were estimated to have the same sensitivity, the last method is characterized by a higher specificity. The results suggest that the use of the UV-vis spectrophotometer increases the specificity of the evaluation of salivary β-galactosidases.