Drug conjugates, in which chemotherapeutic or cytotoxic agents are coupled to targeting or delivering macromolecules like peptides or proteins via a linker, revolutionize cancer treatment. While protein-drug and antibody-drug conjugates have already secured a role in clinical oncology, peptide-drug conjugates (PDCs) are emerging as a promising alternative, offering enhanced efficacy and fewer side effects compared to the free drug molecules. Comprehensive chemical and biological investigation of PDCs is crucial during drug development and optimization, paving the way for the next generation of targeted therapies. Anthracycline-containing peptide conjugates have emerged as promising candidates in targeted cancer therapies due to their ability to deliver cytotoxic agents directly to tumor cells. However, their structural complexity poses significant analytical challenges, particularly in mass spectrometric characterization. Accurate identification and quantification of these conjugates are critical for assessing their stability, efficacy, and mechanism of action. This article explores the major difficulties encountered during mass spectrometry (MS) analysis of anthracycline-peptide conjugates, focusing on ionization issues, fragmentation behavior, and challenges of detection from biological matrix.