Antiphospholipid syndrome (APS) is an autoimmune condition typified by a propensity to thrombosis and pregnancy loss. Dilute Russell’s viper venom testing (DRVVT) is an important cornerstone for APS diagnosis. International guidelines differ in the laboratory protocol for DRVVT, particularly regarding the timing and employment of mixing studies. This study aimed to prospectively compare the result interpretation of the South African (SA) national guidelines to those of the International Society on Thrombosis and Haemostasis (ISTH). Methods: The study included 157 samples submitted for routine lupus anticoagulant (LA) testing to the National Health Laboratory Service (NHLS) at Chris Hani Baragwanath Academic Hospital (CHBAH). Mixing studies were performed on all samples with prolonged screen times (per ISTH protocol). Pertinent clinical and laboratory information was recorded from the laboratory information system. Test positivity rates, sensitivity, and specificity were calculated. Results: Significant differences in the test positivity rates were present between the SA (43/157, 27.4%) and ISTH protocols (18/157, 11.5%; p = 0.0005). The sensitivity of the SA protocol was considerably higher than that of the ISTH protocol (57.1% vs. 28.6%), while the specificity was somewhat lower (78.1% vs. 93.8%). Mixing studies were required significantly more frequently in the ISTH protocol (59.9% vs. 20.4% in the SA protocol; p < 0.001). Conclusion: The ISTH protocol has poorer sensitivity than local guidelines for DRVVT in the SA setting and requires substantially more mixing studies. Therefore, the SA guideline remains the preferred protocol in our laboratory.