Pre-mRNA splicing is an essential step in the expression of most human genes. Mutations at the 5′ splice site (5′ss) frequently cause defective splicing and disease due to interference with the initial recognition of the exon-intron boundary by U1 small nuclear ribonucleoprotein (snRNP), a component of the spliceosome. Here, we use a massively parallel splicing assay (MPSA) in human cells to quantify the activity of all 32,768 unique 5′ss sequences (NNN/GYNNNN) in three different gene contexts. Our results reveal that although splicing efficiency is mostly governed by the 5′ss sequence, there are substantial differences in this efficiency across gene contexts. Among other uses, these MPSA measurements facilitate the prediction of 5′ss sequence variants that are likely to cause aberrant splicing. This approach provides a framework to assess potential pathogenic variants in the human genome and streamline the development of splicing-corrective therapies.