Nisin, a bacteriocin produced by Lactococcus lactis subsp. lactis, is used in some types of food preservation due to its inhibitory action on Gram-positive bacteria and their spores. A commonly used agar diffusion bioassay technique for quantification of nisin in food samples was modified to increase its sensitivity, accuracy and precision. Several variables were evaluated. Results showed Micrococcus luteus as the most sensitive organism tested, a lower agar concentration (0 x 75% compared 1 x 5%) increased the sensitivity of the assay (21% improvement over standard method), and incorporation of 1% Na2HPO4 buffer into the bioassay agar made it possible to prevent false inhibitory zones from developing due to the low pH of the test solutions. This resulted in a 57% improvement in accuracy and a 12% improvement in precision compared to the standard method.