In the last decade, targeting membrane lipids in cancer cells has been a promising approach that deserves attention in the field of anticancer drug development. To get a comprehensive understanding of the effect of the drug [Ru(η⁵-Cp)(PPh₃)₂CN] (RuCN) on cell lipidic components, we combine complementary analytical approaches, matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI TOF MS) and synchrotron radiation-based Fourier transform infrared (SR-FTIR) spectroscopy. Techniques are used for screening the effect of potential metallodrug, RuCN, without and with drug carriers (carbon dots (CDs) and nitrogen-doped carbon dots (N-CDs)) on the lipids of the human ovarian cancer cell line A2780. MALDI TOF MS results revealed that the lysis of ovarian cancer membrane lipids is promoted by RuCN and not by drug carriers (CDs and N-CDs). Furthermore, SR-FTIR results strongly suggested that the phospholipids of cancer cells undergo oxidative stress after the treatment with RuCN that was accompanied by the disordering of the fatty acid chains. On the other hand, using (N-)CDs as RuCN nanocarriers prevented the oxidative stress caused by RuCN but did not prevent the disordering of the fatty acid chain packing. Finally, we demonstrated that RuCN and RuCN/(N-)CDs alter the hydration of the membrane surface in the membrane–water interface region.