Although studies have identified hundreds of loci associated with human traits and diseases, pinpointing causal alleles remains difficult, particularly for non-coding variants. To address this challenge, we adapted the massively parallel reporter assay (MPRA) to identify variants that directly modulate gene expression. We applied it to 32,373 variants from 3,642 cis-expression quantitative trait loci and control regions. Detection by MPRA was strongly correlated with measures of regulatory function. We demonstrate MPRA’s capabilities for pinpointing causal alleles, using it to identify 842 variants showing differential expression between alleles, including 53 well-annotated variants associated with diseases and traits. We investigated one in detail, a risk allele for ankylosing spondylitis, and provide direct evidence of a non-coding variant that alters expression of the prostaglandin EP4 receptor. These results create a resource of concrete leads and illustrate the promise of this approach for comprehensively interrogating how non-coding polymorphism shapes human biology. Display omitted •A new version of MPRA with greater throughput and sensitivity•Evaluation of 32,373 variants associated with eQTLs in lymphoblastoid cell lines•842 variants showed differential gene expression between alleles•Use of CRISPR/cas9 to identify a distal eQTL causal allele for PTGER4 A massively parallel reporter assay analyzes thousands of human polymorphisms to identify alleles that impact gene expression, providing a tool with which to move from disease-associated GWAS hits to the identification of functional variants.