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HDL-mediated cholesterol efflux capacity (CEC) may protect against cardiovascular disease. However, CEC assays are not standardized, hampering their application in large cohorts and comparison between studies. To improve standardization, we systematically investigated technical differences between existing protocols that influence assay performance that have not been previously addressed. CEC was measured in 96-well plates using J774A.1 macrophages labeled with BODIPY-cholesterol and incubated for 4 h with 2% apolipoprotein B-depleted human serum. The time zero method, which calculates CEC using control wells, and the per-well method, which calculates CEC based on the actual content of BODIPY-cholesterol in each well, were compared in 506 samples. We showed that the per-well method had a considerably lower sample rejection rate (4.74% vs. 13.44%) and intra-assay (4.48% vs. 5.28%) and interassay coefficients of variation (two controls: 7.85%, 9.86% vs. 13.58%, 15.29%) compared with the time zero method. Correction for plate-to-plate differences using four controls on each plate also improved assay performance of both methods. In addition, we observed that the lysis reagent used had a significant effect. Compared with cholic acid, lysis with sodium hydroxide results in higher (P = 0.0082) and Triton X-100 in lower (P = 0.0028) CEC values. Furthermore, large cell seeding errors (30% variation) greatly biased CEC for both referencing methods (P < 0.0001) as measured by a resazurin assay. In conclusion, lysis reagents, cell numbers, and assay setup greatly impact the quality and reliability of CEC quantification and should be considered when this method is newly established in a laboratory.