Background HIV enzyme-linked immunosorbent assay (ELISA) is one of the most requested test sets within Virology and forms an essential part of patient management. Assessment of the rejection criteria is a key quality indicator, crucial for improving laboratory services and efficiency to ensure accurate and reliable results.Objectives The aim of this study was to identify the factors that influence the HIV 1/2 serology rejection rates (RR) at Charlotte Maxeke Johannesburg Academic Hospital and to evaluate the associated costs.Methods A retrospective study was conducted (June to December 2019) to identify the RR and rejection criteria of HIV serology samples throughout the total testing process. Descriptive analysis using percentages and frequencies was used to analyse the RR by phase, health establishment, ward and healthcare professional. A cost analysis incorporating minor and major costs was modelled in each phase of testing, and the total cost of rejections was calculated.Results A total of 6678 tests were received, and 738 were rejected (RR = 11.1%). The pre-analytical phase contributed significantly to the overall RR, with the requirement of a separate sample (57.44%) the most common reason for rejection. The total cost per rejected test was $2.47, which amounted to a total rejection cost of $197.55, of which $158.18 was caused by the pre-analytical rejection criteria.Conclusion High RR of HIV tests were noted, resulting in significant cost wastage. Identification and analysis of rejections must be implemented across all laboratories to improve the efficiency of testing, provide a cost-saving benefit and maintain high laboratory standards.