Untargeted lipidomic samples are extremely complex and often exceed the limits of peak capacity achievable by one-dimensional liquid chromatography (LC). Comprehensive two-dimensional liquid chromatography (LC × LC) appears as a promising alternative to overcome this drawback. Unfortunately, this approach generates highly complex datasets which untargeted analysis is challenging. In this work, a global methodological strategy combining LC × LC-MS/MS with chemometric data analysis is proposed for untargeted lipidomic studies. The feasibility of the proposed methodology is demonstrated by its application to assess the effects of arsenic exposure on the lipidome of growing rice samples. A two-dimensional chromatographic setup coupling reversed phase (RP) and hydrophilic interaction liquid chromatography (HILIC) modes together with a triple quadrupole mass detector (TQD) is proposed to analyze lipid extracts from rice samples at different experimental conditions. Chemometric tools were used for data compression, spectral and elution profiles resolution, feature detection and statistical analysis of the multidimensional LC × LC-MS/MS data. The obtained results revealed that the proposed methodology was useful to gather relevant information from untargeted lipidomic studies and detect potential biomarkers.