A simple and inexpensive fluorescence-based technique has been developed for high-throughput antimalarial drug screening. This method uses SYBR Green I, a dye that enhances fluorescence in the presence of DNA, to measure parasite growth. It was compared to a standard radioisotopic method using Plasmodium falciparum strain D6. Both methods determined the effective concentration (EC50) of drugs that caused a 50% reduction in parasite counts after 48 hours. The EC50 values for various antimalarial drugs were similar or identical using both techniques, indicating the fluorescence method's reliability. The fluorescence assay is simpler, cheaper, and faster than the radioisotopic method, making it suitable for high-throughput screening. It uses a lysis buffer containing saponin and detergent to facilitate DNA detection. The method is robust, applicable to automated analysis, and does not require radioactive materials or complex procedures. The EC50 values were not significantly affected by a freeze-thaw cycle, suggesting the method's stability. The fluorescence assay showed a linear relationship between parasitemia levels and fluorescence units, with a high correlation coefficient (r² = 0.9763). It is less sensitive to variations in experimental conditions compared to the radioisotopic method, which requires strict controls. The method is adaptable to various conditions, including different parasite strains and culture settings. It has been successfully used in our laboratory to replace radioisotopic assays, performing hundreds of tests under diverse conditions. SYBR Green I is a sensitive DNA stain, more sensitive than ethidium bromide. The lysis buffer was formulated to enhance fluorescence detection without requiring a freeze-thaw cycle. The method is safe, with SYBR Green I being less mutagenic than ethidium bromide. It is suitable for field settings where radioisotope use is impractical. The fluorescence-based assay offers a cost-effective, simple, and reliable alternative to radioisotopic methods for antimalarial drug screening. Further studies are needed to optimize the method and confirm its applicability to a wide range of drugs and conditions. The method is broadly applicable and has shown promise in replacing traditional techniques for high-throughput screening.A simple and inexpensive fluorescence-based technique has been developed for high-throughput antimalarial drug screening. This method uses SYBR Green I, a dye that enhances fluorescence in the presence of DNA, to measure parasite growth. It was compared to a standard radioisotopic method using Plasmodium falciparum strain D6. Both methods determined the effective concentration (EC50) of drugs that caused a 50% reduction in parasite counts after 48 hours. The EC50 values for various antimalarial drugs were similar or identical using both techniques, indicating the fluorescence method's reliability. The fluorescence assay is simpler, cheaper, and faster than the radioisotopic method, making it suitable for high-throughput screening. It uses a lysis buffer containing saponin and detergent to facilitate DNA detection. The method is robust, applicable to automated analysis, and does not require radioactive materials or complex procedures. The EC50 values were not significantly affected by a freeze-thaw cycle, suggesting the method's stability. The fluorescence assay showed a linear relationship between parasitemia levels and fluorescence units, with a high correlation coefficient (r² = 0.9763). It is less sensitive to variations in experimental conditions compared to the radioisotopic method, which requires strict controls. The method is adaptable to various conditions, including different parasite strains and culture settings. It has been successfully used in our laboratory to replace radioisotopic assays, performing hundreds of tests under diverse conditions. SYBR Green I is a sensitive DNA stain, more sensitive than ethidium bromide. The lysis buffer was formulated to enhance fluorescence detection without requiring a freeze-thaw cycle. The method is safe, with SYBR Green I being less mutagenic than ethidium bromide. It is suitable for field settings where radioisotope use is impractical. The fluorescence-based assay offers a cost-effective, simple, and reliable alternative to radioisotopic methods for antimalarial drug screening. Further studies are needed to optimize the method and confirm its applicability to a wide range of drugs and conditions. The method is broadly applicable and has shown promise in replacing traditional techniques for high-throughput screening.