During the various stages of the clinical and process development, potency assays are a critical part of any biologics. Potency assays are linking manufacturing with the clinics by translating biochemical properties and stability of a molecule with its biological activity. Sometimes it may become necessary to replace an existing potency method with an improved or new method. Through early phase of nimotuzumab development process the bioassay was based upon mAb binding to a cell-surface expressed antigen on viable cells and assessed via flow cytometry. For late phases, a new relative potency assay was developed and validated, according to USP and ICH guidelines. The new bioassay is based on the inhibition of NCI-H292 cell proliferation in response to nimotuzumab and was validated for its specificity, relative accuracy (dilutional linearity study), precision (repeatability & Inter run variability), range and robustness. The bioassay demonstrated to be robust, sensitive, leading a specific and good linearity (coefficient of determination of 0.9732), precise with coefficients of variation (CV) less than 11% for both repeatability and intermediate precision. Sample recovery studies demonstrated the bioassay is accurate and linear from 50 to 150%. The assay was also stability-indicating since it was able to detect changes in nimotuzumab degraded samples and can better reflect the quality fluctuation of different batches. The new bioassay requires level of receptor occupancy and target turnover, two key components for further understanding the link between clinical drug exposure and in vitro potency.