Methionine restriction selectively arrests cancer cells during the S-phase of the cell cycle. We hypothesized that DNA damage may occur in S-phase in cancer cells during methionine restriction. To determine if this occurs, we used MiaPaCa-2Tet-On 53BP1-green fluorescent protein (GFP) pancreatic cancer cells, which report GFP fluorescence in real time after DNA-damage response (DDR) in these cells. We also determined whether a chemotherapy drug in combination with methionine restriction increases the rate of DNA damage. MiaPaCa-2Tet-On 53BP1-GFP cells were used for in vitro experiments. The 25% and 50% inhibitory concentrations (IC25 and IC50, respectively) of recombinant methioninase (rMETase) and paclitaxel on MiaPaCa-2Tet-On 53BP1-GFP pancreatic cancer cells were determined. Cell viability and DDR with rMETase alone, paclitaxel alone, and their combination were measured in MiaPaCa-2Tet-On 53BP1-GFP cells. The IC25 of rMETase on MiaPaCa-2Tet-On 53BP1-GFP cells was 1.66 U/ml. The IC25 for paclitaxel on MiaPaCa-2Tet-On 53BP1-GFP cells was 3.31 nM. The combination of rMETase and paclitaxel synergistically reduced the viability of MiaPaCa-2Tet-On 53BP1-GFP cells. The IC50 of paclitacel on MiaPaCa-2Tet-On 53BP1-GFP cells was 5.1 nM. The IC50 of rMETase on MiaPaCa-2Tet-On 53BP1-GFP cells was 2.3 U/ml. The combination of rMETase (IC50) plus paclitaxel (IC50) on MiaPaCa-2Tet-On 53BP1-GFP cells also caused more DNA damage than either agent alone. The present study suggests the synergy of methionine restriction and chemotherapy is due, at least in part, to DNA damage of cancer cells.