Rituximab, a chimeric monoclonal antibody targeting CD20, is widely used in the treatment of various malignancies and autoimmune disorders. In this study, an ELISA was developed to quantitate the rituximab levels, using mouse anti-rituximab (CDR specific) antibody as capture antibody. Accurate pharmacokinetic (PK) monitoring is essential for optimizing dosing regimens and improving therapeutic outcomes. Initially, surface plasma resonance showed acceptable binding kinetics (Kd) of 0.5x10-09 for rituximab and non-cross reactive to human IgG. A standard curve of rituximab (1.56ng/ml to 800ng/ml), with five quality control points (6.25, 18.75, 25, 150, and 200ng/ml) was evaluated for inter and intra-assay accuracy and precision, using serum matrix, by four independent validations. The linear range of the validated assay was 1.56ng/ml to 800ng/ml, and ULOQ and LLOQ were 200ng/ml and 6.25ng/ml, respectively. The assay specificity was validated by testing cross-reactivity of rituximab with adalimumab, which found to be non reactive. Further, the hook effect was over-ruled by diluting the highest concentration of rituximab tested to assay linear range, and dilution integrity was observed for the entire concentrations within linear range (%RE ≤20%), as recommended by European Medicines Agency. Collectively, this binding-based ELISA method is highly sensitive, reproducible, and useful for monitoring rituximab.