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mRNA therapeutics offer a potentially universal strategy for the efficient development and delivery of therapeutic proteins. Current mRNA vaccines include chemically modified nucleotides to reduce cellular immunogenicity. Here, we develop an efficient, high-throughput method to measure human translation initiation on therapeutically modified as well as endogenous RNAs. Using systems-level biochemistry, we quantify ribosome recruitment to tens of thousands of human 5′ untranslated regions (UTRs) including alternative isoforms and identify sequences that mediate 200-fold effects. We observe widespread effects of coding sequences on translation initiation and identify small regulatory elements of 3–6 nucleotides that are sufficient to potently affect translational output. Incorporation of N1-methylpseudouridine (m1Ψ) selectively enhances translation by specific 5′ UTRs that we demonstrate surpass those of current mRNA vaccines. Our approach is broadly applicable to dissecting mechanisms of human translation initiation and engineering more potent therapeutic mRNAs.