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In this study, spores of B. cereus NCDC 66 producing β-lactamase enzyme through induction, was used as indicator. Dextrose (200 mg) and whey powder (230 mg) were added to milk and incubation at 37ºC for 5.30 h for germination and induction. Enzyme induction was quantified using modified iodometric test after induction with penicillin G spiked milk samples as per Codex recommended MRL. The results were compared with AOAC approved Charm 6602 microbial receptor assay and microbial inhibition test kit. The application of developed assay on 75 random milk samples involving raw, packaged pasteurized and dried milks revealed 4 of raw milk and 1 of packaged pasteurized milk found to be positive for β-lactam antibiotic. The assays results were in complete correlation with reference methods used. The specificity of the assay was confirmed by absence of induction in experiment with sample containing non β-lactam group. Novel β-lactamase induction based principle can be applied for specific detection of β-lactam antibiotic residues in dairy foods.