Purpose This study was meant to determine the effect of time to plasma separation, storage duration, freeze-thawing cycle and dilution proportion on the HIV-1 viral load level. Methods Experimental study design was employed by collecting 10mL whole blood samples into two EDTA tubes from 88 eligible HIV infected patients at St Paul’s Hospital Millennium Medical College. The viral load test was done using Abbott m2000sp/rt analyzer. Data was entered into Microsoft excel and analyzed by SPSS version 20. Repeated measure analysis of variance was used to compare HIV RNA viral load mean difference between different time to plasma separation, storage, freeze-thawing cycles and dilution levels. Post-hoc analysis was employed to locate the place of significant differences. P value less than 0.05 was used to declare statistical significance while viral RNA level of 0.5 log copies/ml was used to determine clinical significance. Results There was significant HIV-1 RNA viral load log mean difference between plasma separation time at 6 hours (hrs) and 24hrs (p<0.001). There was also significant HIV-1 RNA viral load log mean difference between plasma tested within 6hrs and those stored at 2–8°C for 15 days (p = 0.006), and between plasma stored at 2–8°C for 6 days versus 15 days (p<0.001). There was significant log mean difference between plasma that was exposed to fourth cycle of freeze-thawing after storage at -20°C when compared with plasma tested within 6hrs (p = 0.013). Conclusion Plasma separated at 24hrs, stored at 2–8°C for 15 days or freeze-thawed for four cycles had significant effect on HIV viral load level. However, the differences were not clinically significant at a cut-off viral load level of 0.5 log copies/ml. Avoiding delays to plasma separation beyond 24 hrs, storing at 2–8°C for 15 days and freeze-thawing for no more than 4 cycles is recommended to improve the result quality.