Hypoxic and nutritionally deprived areas are thought to contribute to resistance of tumour cells to apoptosis in pancreatic cancers. Tumour M2-pyruvate kinase, a tumour associated isoenzyme of pyruvate kinase, is elevated in patients with pancreatic cancer. This study aims to measure tumour M2-pyruvate kinase (TuM2-PK) in different human pancreatic cancer cell line following exposure to acidic pH, glucose deprived and hypoxic conditions. TuM2-PK expression was measured in cell lysate supernatant of HMEF, MCF7, Panc 1 and Colo357 human cell lines using sandwich type ELISA (ScheBo® Tumour M2-PK™) and Western blot. Panc1 and Colo 357 were grown in acidic pH (6.5), glucose free medium (DMEM)and hypoxic (1% Oxygen) condition. TuM2-PK was expressed as mUnits/million cells. Mean TuM2-PK values between different cell lines were compared using two-way ANOVA and unpaired T test. Correlation between TuM2-PK levels and cell densities was made using Pearson’s coefficient. M2-PK expression was significantly higher in Colo357 cells compared to HMEF, MCF 7 (p<0.05) and Panc 1 cells (p<0.001). Correlation between TuM2-PK levels and different cell densities was negative[MCF7 (r=-0.382, p=0.06), Panc-1 (r=-0.096, p=0.065) and Colo357 (r-0.528, p<0.01). Tumour M2-PK expression was significantly enhanced in Colo357 cells (p<0.05) at acidic pH or glucose deprived or hypoxic condition in the culture medium compared to normal conditions. TuM2-PK can be measured in different cell lines using ELISA. In-vitro expression of TuM2-PK correlates with low cell density. Acidic, glucose deprived and hypoxic conditions around tumour cells enhances M2-PK expression.