Ensuring the quality and performance of human immunodeficiency virus, type 1 (HIV-1) enzyme-linked immunosorbent assay (ELISA) testing in routine laboratory situations is an important practical concern. In this article we develop statistical quality control procedures designed to monitor test performance of the microplates being processed. First, the logarithm of measured optical density is shown to be a more appropriate scale on which to work. Shewhart control charts are considered but are not entirely satisfactory because of between-plate variation. Correct classification depends more on within-plate variation than on between-plate variation. Range charts are useful, but they do not directly indicate whether the results from any particular microplate are reliable. Consequently, a new statistical control chart—the separation chart—is proposed, and the assumptions on which it is based are empirically verified. Retrospective analysis of nearly 1,300 microplates, using separation and range charts, demonstrates that these statistical process control methods may represent a potentially useful supplement to the manufacturer's recommended quality control procedures.