Background. Unavailability of accurate, rapid, reliable, and cost-effective malaria diagnostic instruments constitutes major a challenge to malaria elimination. We validated alternative malaria diagnostic instruments and assessed their comparative cost-effectiveness. Method. Using a cross-sectional study design, 502 patients with malaria symptoms at selected health facilities in Ibadan between January and April 2014 were recruited consecutively. We examined malaria parasites using Cyscope®, QBC, and CareStart™ and results were compared to light microscopy (LM). Validity was determined by assessing sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV). Costs per hour of use for instruments and turnaround time were determined. Result. Sensitivity of the instruments was 76.0% (CareStart), 95.0% (Cyscope), and 98.1% (QBC). Specificity was 96.0% (CareStart), 87.3% (Cyscope), and 85.5% (QBC). PPV were 65.2%, 67.5%, and 84.7%, while NPV were 93.6%, 98.6%, and 99.4% for CareStart, Cyscope, and QBC with Kappa values of 0.75 (CI = 0.68-0.82) for CareStart, 0.72 (CI = 0.65-0.78) for Cyscope, and 0.71 (CI = 0.64-0.77) for QBC. Average cost per hour of use was the lowest ($2.04) with the Cyscope. Turnaround time was the fastest with Cyscope (5 minutes). Conclusion. Cyscope fluorescent microscope had the shortest turnaround time and is the most cost-effective of all the malaria diagnostic instruments evaluated.