In this study, we wanted to examine the effects of 5-fluorouracil, a drug used in anticancer treatment, on the proteome of HEK293T cells (human cell culture). HEK293T cells were exposed to 5-fluorouracil (5-FU) using two different incubation strategies. In the first experiment, cells were incubated in 5-FU supplied medium and harvested after 24h acute exposure. The second experiment included a 24h exposure to 5-FU, followed by a recovery phase, were cells were incubated for another 6h in fresh medium without 5-FU. We performed shotgun proteomics of the respective tryptic digest of both experiments and compared the proteome of treated and untreated cells, respectively.