Ki67, a nuclear proliferation-related protein, is heavily used in anatomic pathology but has not become a companion diagnostic or a standard-of-care biomarker due to analytic variability in both assay protocols and interpretation. The International Ki67 Working Group in breast cancer has published and has ongoing efforts in the standardization of the interpretation of Ki67, but they have not yet assessed technical issues of assay production representing multiple sources of variation, including antibody clones, antibody formats, staining platforms, and operators. The goal of this work is to address these issues with a new standardization tool. We have developed a cell line microarray system in which mixes of human Karpas 299 or Jurkat cells (Ki67+) with Sf9 (Spodoptera frugiperda) (Ki67-) cells are present in incremental standardized ratios. To validate the tool, six different antibodies, including both ready-to-use and concentrate formats from six vendors, were used to measure Ki67 proliferation indices using IHC protocols for manual (bench-top) and automated platforms. The assays were performed by three different laboratories at Yale and analyzed using two image analysis software packages, including QuPath and Visiopharm. Results showed statistically significant differences in Ki67 reactivity between each antibody clone. However, subsets of Ki67 assays using three clones performed in three different labs show no significant differences. This work shows the need for analytic standardization of the Ki67 assay and provides a new tool to do so. We show here how a cell line standardization system can be used to normalize the staining variability in proliferation indices between different antibody clones in a triple negative breast cancer cohort. We believe that this cell line standardization array has the potential to improve reproducibility among Ki67 assays and laboratories, which is critical for establishing Ki67 as a standard-of-care assay.