N6-methyladenosine (m6A) is the most abundant modification on mRNA and is implicated in critical roles in development, physiology, and disease. A major limitation has been the inability to quantify m6A stoichiometry and the lack of antibody-independent methodologies for interrogating m6A. Here, we develop MAZTER-seq for systematic quantitative profiling of m6A at single-nucleotide resolution at 16%–25% of expressed sites, building on differential cleavage by an RNase. MAZTER-seq permits validation and de novo discovery of m6A sites, calibration of the performance of antibody-based approaches, and quantitative tracking of m6A dynamics in yeast gametogenesis and mammalian differentiation. We discover that m6A stoichiometry is "hard coded" in cis via a simple and predictable code, accounting for 33%–46% of the variability in methylation levels and allowing accurate prediction of m6A loss and acquisition events across evolution. MAZTER-seq allows quantitative investigation of m6A regulation in subcellular fractions, diverse cell types, and disease states. • RNA digestion via m6A sensitive RNase (MAZTER-seq) allows systematic m6A quantitation • MAZTER-seq reveals that antibody-based methods are of limited sensitivity • m6A stoichiometry is "hard coded" by a simple, predictable, and conserved code • MAZTER-seq allows quantitative tracking of m6A in diverse biological settings A new enzymatic approach for precise mapping and measurement of m6A within mRNAs provides insight into how methylation sites are selected and the functional impact of the modifications. [ABSTRACT FROM AUTHOR]