Cytochrome c (cytc) is a heme protein of 12 kDa that transfers electrons in the mitochondrial respiratory chain. Increased cytc peroxidase activity leads to cardiolipin (CL) oxidation, a hallmark of early apoptosis stage. Here, we aimed to investigate the interaction between cytc with cardiolipin hydroperoxide (CLOOH) in a mimetic mitochondrial membrane. Cytc-CL peroxidase reaction occurred at faster rates with CLOOH than with H2O2. Moreover, liposomes containing CLOOH promoted increased protein aggregation with minor or no release of cytc from the membrane. Dimeric and trimeric cytc species were observed in the first 15 min, followed by increased formation of high-molecular-weight aggregates afterwards. nLC-MS/MS analysis identified several Lys and His residues covalently modified by lipid aldehydes that showed mass increments corresponding to 4-hydroxynonenal (HNE), 4-oxononenal (ONE), hexanoyl, heptenal and octenal addition. Noteworthy, most modifications were observed at Lys and His residues located at A-site (K73, K87, K88), L-site (H26, H33, and K27) membrane binding sites. Further, dityrosine cross-linked peptides were also characterized at residues Y48-Y74, Y48-Y97 and Y74-Y97. Collectively, our findings show that CLOOH causes irreversible protein damage and crosslinking of cytc in the membrane. •Cytc reacts with cardiolipin hydroperoxides at faster rates than with hydrogen peroxide.•Cytc binds to liposomes containing cardiolipin hydroperoxides and becomes highly aggregated.•Lys and His residues involved in cytc-CL electrostatic binding sites are modified by aldehydes.•Tyr residues located at cytc surface are involved in dityrosine crosslinking.