The aim of this work was to develop a rapid resolution liquid chromatography coupled to electrospray ionization time-of-flight mass spectrometry (RRLC-ESI-TOF-MS) method followed by tetrazolium salt (MTT)-based cell viability assays for qualitative and quantitative classification of extra virgin olive oil (EVOO) varieties by phenolic and other polar compound contents as well as for rapid characterization of putative cytotoxic activities against human cancer cells. Five different Spanish EVOO varieties were analyzed, and RRLC-ESI-TOF-MS method was applied for qualitative and quantitative identification of most important phenolic compounds. We finally employed MTT-based cell viability protocol to assess the effects of crude EVOO phenolic extracts (PEs) on the metabolic status of cultured SKBR3 human breast cancer cells. MTT-based cell viability assays revealed a wide range of breast cancer cytotoxic potencies among individual crude PE obtained from EVOO monovarietals. Remarkably, breast cancer cell sensitivity to crude EVOO-PEs was up to 12 times higher in secoiridoids enriched-PE than in secoiridoids-low/null EVOO-PE.