We have observed that the serum levels of certain metabolites differ between Chilean patients with gallbladder cancer and those with gallstone disease. Based on these discovery results, we selected ten candidate metabolites based on the smallest p-values among metabolites exhibiting a fold-change greater than 1.5. The aim of the present study is to validate these ten differential metabolite levels in an independent cohort of Chilean patients with gallbladder cancer and gallstone disease, and to assess the transferability of these findings to other patient populations.