It is widely believed that to facilitate growth and uncontrolled proliferation, cancer cells undergo metabolic reprogramming which includes upregulation of de novo lipogenesis and protein synthesis. Using deuterated water (2H2O), a stable isotope metabolic tracer, it is possible to reliably label multiple cellular biomass components (lipids, protein and DNA), thus permitting measurement of their synthesis. We have used this approach to characterise the turnover rates and metabolism of a panel of human colon cancer cell lines in vitro and in solid mouse tumours in vivo using the DEN-induced hepatocellular carcinoma (HCC) model. Our in vitro data show that in the colon cancer cells, biomass synthesis rates were strongly linked to the rate of cell division, with rapidly dividing cells having greater lipid and protein synthetic rates. Furthermore, the cells displayed a marked heterogeneity in the reliance on the de novo lipogenic pathway, with dependence on both ‘self-made’ and exogenously-derived fatty acid. In the in vivo setting, DEN-treated mice were administered 2H2O 20 h prior to collection of both tumour and adjacent normal liver tissue. Surprisingly, tumours did not have heightened rates of de novo lipogenesis. Tumour fractional protein synthetic rates were elevated by ~15%; however, tumour tissues had ~20% lower protein pool size (likely due to anaplasia) resulting in a ~10% decrease in absolute protein synthetic rates compared to non-tumour tissue. DNA synthesis could not be detected in either tumour or adjacent normal tissue over the 20 h labelling period. These results suggest that in its late stages, DEN-induced tumours do not display increased lipogenesis.