High-throughput assay systems have had a disproportionally large impact on uncovering how cells function, as well as how misregulation can lead to disease. However, no high-throughput assay systems have been developed to systematically address how mutations impact molecular functions or cell processes in human cells. This is arguably one of the most critical assays because human pathology and treatment are largely based upon molecular functions. To address this challenge, herein we engineered, developed, and tested the first modular high-throughput molecular function assay system. Note that this is not a selection lethality screen! This “GigaAssay” single cell / one-pot assay system was adapted to study how variants impact HIV Tat-driven transactivation of a green fluorescent protein (GFP) reporter. We assayed all 1,615 Tat single and 3,429 double amino acid substitutions with no single mutant dropout. Each mutant was assayed with replicate observations in LentiX293T and Jurkat cells with an average of 100s of separately barcoded cDNA molecules and cell groups for each mutant. Each mutant had ~2,000X-90,000X sequencing coverage to measure its transcriptional activity and had p value ranging as low as 10-271. Five independent assay performance assessments with benchmark data, individually tested clones, and replicate comparisons all indicate exceptional reproducibility, accuracy, and robustness. The shortcomings of alanine scanning mutagenesis and protein truncation studies are revealed by including exhaustive substitution tolerance and intragenic epistasis in the typical structure/function analysis(structure/function/tolerance/epistasis). This flexible and extensible technology enables a far more comprehensive holistic view of protein molecular function and yet with a highly simplified single-pot assay.