CA125 is heavily relied upon for diagnosis and prognosis of epithelial ovarian cancer; however the molecular characteristics are poorly understood and are the subject of ongoing investigations. This review summarizes and evaluates the available data regarding the molecular features of CA125. While the nucleotide sequence of this protein was independently cloned by two groups and showed an almost complete homology between the overlapping parts, other molecular features like predominant type of glycosylation and isoforms are not only disputed but mutually exclusive. Although mass spectrometry has already been shown to be a valid option to clarify these ambiguous findings, most data generated by this method, and published so far, does not satisfy established reporting standards. This review highlights the importance of high quality mass spectrometric data to independently verify the identity of the investigated protein. This would ensure that CA125 identified by antibody-dependent methods are not cross-reactive proteins. Therefore, the ambiguous views on the glycan structure as well as the controversial low molecular weight forms can be clarified. This greater understanding may help improve established measurement techniques for assaying CA125 serum levels. Alternatively, novel detection systems that are independent of antibodies, such as selective reaction monitoring mass spectrometry (SRM), could also be applied for CA125 quantitation in patient samples. SRM has already been shown to be a feasible approach to quantify proteins in plasma [61]. Furthermore, detailed structural information on CA125 will enable the development of new and more refined molecularly targeted antibody therapies which already show great clinical potential and warrant further research [62–65].