Next generation sequencing (NGS) of biopsy-extracted material remains the gold standard for tumor profiling. Panels designed for this type of testing often target 500+ genes, creating a challenge for analytic validation in a clinical setting. Use of patient-like multiplexed commercial reference materials can be a more cost effective and reliable option versus relying on remnant samples with mutations of interest, especially with more stringent requirements from the FDA and other regulators in the early stages of implementation. Here we describe the development and multisite evaluation of expanded Seraseq Tumor Mutation DNA Mix v3 reference materials, which contain an expanded variant list to meet the growing needs of NGS tumor profiling assay developers.