Translesion DNA synthesis (TLS) is a DNA damage tolerance mechanism, in which specialized low-fidelity DNA polymerases bypass lesions that interfere with replication. This process is inherently mutagenic due to the miscoding nature of DNA lesions, but it prevents double strand breaks, genome instability, and cancer. We describe here a quantitative method for measuring TLS in mammalian cells, based on non-replicating plasmids that carry a defined and site-specific DNA lesion in a single-stranded DNA region opposite a gap. The assay is responsive to the cellular composition of TLS DNA polymerases, and TLS regulators. It can be used with a broad variety of cultured mammalian cells, and is amenable to RNAi gene silencing, making it a useful tool in the study of TLS in mammalian cells.