Ceramic hydroxyapatite (CHT) high-performance liquid chromatography (HPLC) is used to purify a variety of classes of monoclonal antibodies (mAbs) from crude murine ascites fluids. We report here that this method is also applicable for simple and efficient purification of many mAb fragments that are generated by pepsin treatment of crude ascites. F(ab′)2 fragments were quantitatively generated from IgG1 mAbs in ascitic fluids by incubation with pepsin for 6 h at pH 3.9–4.1. Under the same conditions, pepsin also cleaved unwanted ascites components, such as albumin and transferrin to very low molecular weight polypeptides. The F(ab′)2 fragments, but not the low molecular weight products, selectively bound to and were eluted from the CHT column using a linear gradient of phosphate ion concentration over 15 min. The recovery of the F(ab′)2 fragments by CHT-HPLC was >90%. This method also allowed single-step purification of mAb fragments from distinct IgG subclasses (IgG2a and IgG2b) and IgM directly from crude digested ascitic samples. This CHT-HPLC method combined with direct pepsinolysis of murine ascites is a useful strategy for rapid purification and characterization of many types of mAb fragments.