We used erythrocytes as the model tissue to evaluate an optimal solution for the extraction of intracellular metabolites and time-dependent variation of the metabolome in living cells. Projection to latent structure (PLS) of the GC/MS and LC/MS data suggested that the most efficient solution for the extraction of metabolites from wet erythrocytes (50 mg) could be a methanol-chloroform-water mixture (950 microL, 700:200:50, v/v/v). PLS-discriminant analysis (DA) clearly profiled a time-dependent alternation of metabolic phenotype of erythrocytes. Identification of the metabolites showed that the process was characterized by accumulating of metabolic products and depleting of nutritious substances in erythrocytes during incubation.