Translesion DNA synthesis (TLS) is an evolutionarily conserved process that cells activate to tolerate DNA damage. TLS facilitates proliferation under DNA damage conditions and is exploited by cancer cells to gain therapy resistance. It has been so far challenging to analyze endogenous TLS factors such as PCNAmUb and TLS DNA polymerases in single mammalian cells due to a lack of suitable detection tools. We have adapted a flow cytometry-based quantitative method allowing detection of endogenous, chromatin-bound TLS factors in single mammalian cells, either untreated or exposed to DNA-damaging agents. This high-throughput procedure is quantitative, accurate, and allows unbiased analysis of TLS factors' recruitment to chromatin, as well as occurrence of DNA lesions with respect to the cell cycle. We also demonstrate detection of endogenous TLS factors by immunofluorescence microscopy and provide insights into TLS dynamics upon DNA replication forks stalled by UV-C-induced DNA damage.