Rituximab (RTX) is a monoclonal antibody which targets CD20 and is approved for treatment of non-Hodgkin's lymphoma (NHL), with an approximate 50% overall response rate among NHL patients. Accurate determination of RTX concentrations in patient plasmas is important for proper dosing of patients and for correlating RTX concentrations with clinical responses. There is currently no assay available for RTX which utilizes easily obtainable commercial reagents. Therefore, we sought to develop such an assay, and in this report we describe three new assays for RTX concentration. One assay, based on flow cytometry, quantitates immunologically active RTX based on its ability to bind to CD20 on Raji cells. Two other methods, based on flow cytometry and ELISA, measure RTX based on its antigenic properties. The assays are accurate, in good agreement with one another, and can all measure RTX concentrations as low as approximately 1 microg/ml in both sera and plasmas. Use of these assays reveals that chronic lymphocytic leukemia (CLL) patients receiving RTX treatment have lower plasma RTX concentrations than patients with other B cell lymphomas at all times over the usual 4-week course of therapy. The level in CLL plasmas often declines to <1 microg/ml RTX 1 week after each RTX infusion, substantially lower than the values found in comparable non-CLL patient plasmas. RTX assay results also demonstrate that naïve CLL patient plasmas do not have levels of non-cell associated CD20 sufficient to either interfere with an in vitro assay of RTX or to block the potential therapeutic action of RTX in vivo.