The external quality assessment schemes (EQAS) organization provides a suitable program to monitor and improve the performance of human immunodeficiency virus (HIV) testing laboratories. It is essential to prepare a EQAS panel using samples with different characteristics by standardized preparation systems, and to correctly assess the results choosing the reasonable analytical methods for EQAS program. The aims of this study are to develop EQAS materials via evaluation of matrix effect on EQAS results and to develop analytical tools for HIV serological test.EQAS panels were prepared by standardized preparation systems. EQAS results, which were performed 5 times in total in 350 clinical laboratories during three-year period, were used to evaluate the matrix effects (w/wo defibination process and sample dilution). Analytical tools applied to serological EQAS results using robust statistics and Youden plots are currently being used for analyzing the EQAS results for chemistry laboratories. The current status of the quality management systems at HIV laboratories in Korea could be identified using a questionnaire.The total number of HIV screening tests performed in a year is approximately 5.5 million. Equipment and HIV testing methods varied by HIV screening laboratories, and most laboratories participated in evaluation programs to manage quality of their HIV testing. The overall performance of clinical laboratories was shown to be enhanced when using unrecalcified plasma panels compared with recalcified panels (p < 0.001). Some EIA kits showed limited detection capability in weakly-positive EQAS material. The traditional method detected 5 laboratories as outliers, while the robust statistics method detected 13 laboratories as outliers for HIV testing showing better discrimination. The outliers detected by the Youden plot were differentiated as six systematic and two random errors. This study suggested that it is possible to use plasma to prepare EQAS panel without defibrination process. Applying an advanced analytical method to serology EQAS is needed to assess laboratory performance such as robust statistics and Youden plots. Therefore, when designing a reliable EQAS, use of HIV panels with different characteristics and advanced statistical methods for data analysis should be considered. This is the first approach to evaluate matrix effects on EQAS results using large-sized data and to apply analytical tools used in the field of chemistry to serological tests.