Background HIV viral load (VL) monitoring informs antiretroviral therapy failure and helps to guide regimen changes. Typically, VL monitoring is performed using dried blood spot (DBS) samples transported and tested in a centralized laboratory. Novel sample collection technologies based on dried plasma stored on a plasma separation card (PSC) have become available. The cost-effectiveness of these different testing approaches to monitor VL is uncertain, especially in resource-limited settings. The objective of this study is to evaluate the potential cost-effectiveness of HIV VL testing approaches with PSC samples compared to DBS samples in Malawi. Methods We developed a decision-tree model to evaluate the cost-effectiveness of two different sample collection and testing methods—DBS and PSC samples transported and tested at central laboratories. The analysis used data from the published literature and was performed from the Malawi Ministry of Health perspective. We estimated costs of sample collection, transportation, and testing. The primary clinical outcome was test accuracy (proportion of patients correctly classified with or without treatment failure). Sensitivity analysis was performed to assess the robustness of results. Results The estimated test accuracy for a DBS testing approach was 87.5% compared to 97.4% for an approach with PSC. The estimated total cost per patient of a DBS testing approach was $19.39 compared to $17.73 for a PSC approach. Based on this, a PSC-based testing approach “dominates” a DBS-based testing approach (i.e., lower cost and higher accuracy). Conclusion The base-case analysis shows that a testing approach using PSC sample is less costly and more accurate (correctly classifies more patients with or without treatment failure) than with a DBS approach. Our study suggests that a PSC testing approach is likely an optimal strategy for routine HIV VL monitoring in Malawi. However, given the limited data regarding sample viability, additional real-world data are needed to validate the results.