BACKGROUND: The complement system plays an important role in the identification and removal of foreign substances and immune complexes and in the stimulation of inflammatory response. The standard 50% hemolytic complement (CH50) assay is the most conventional method for functional activity of the classical complement pathway but it has been avoided in clinical laboratories because of labor intensive procedure and lack of standard protocol. This study was carried out to set up and evaluate the conventional CH50 test using in-house reagents including sensitized sheep erythrocytes and standard sera. METHODS: CH50 assay was performed according to the modified Mayor's method. The linearity, within-run and between-run precision was evaluated. RESULTS: The within run coefficient of variation (CV) was less than 5% and between run CV was 16.7%, which was improved to 2.8% when in-house standard sera was used as calibrator in each test. The linearity was well preserved (R2 = 0.9993). CONCLUSIONS: CH50 test showed excellent precision and linearity with use of in-house standard sera. It would be very useful for the evaluation of classical complement pathway activity in clinical immunology laboratory.