This review paper discusses various methods for evaluating antimicrobial activity in vitro, focusing on their advantages and limitations. The methods reviewed include diffusion techniques such as disk-diffusion, agar gradient (Etest), well diffusion, plug diffusion, cross streak, and poisoned food methods. Thin-layer chromatography (TLC)–bioautography, which combines TLC with biological and chemical detection methods, is also discussed. Dilution methods, such as broth and agar dilution, are highlighted for their ability to determine minimum inhibitory concentrations (MICs). The time-kill test and ATP bioluminescence assay are introduced for their rapid and quantitative results, while flow cytometry is noted for its ability to detect antimicrobial resistance and cell damage but is limited by the availability of specialized equipment. The paper emphasizes the importance of standardized protocols and methodological adaptations when testing natural products to ensure accurate and reproducible results.This review paper discusses various methods for evaluating antimicrobial activity in vitro, focusing on their advantages and limitations. The methods reviewed include diffusion techniques such as disk-diffusion, agar gradient (Etest), well diffusion, plug diffusion, cross streak, and poisoned food methods. Thin-layer chromatography (TLC)–bioautography, which combines TLC with biological and chemical detection methods, is also discussed. Dilution methods, such as broth and agar dilution, are highlighted for their ability to determine minimum inhibitory concentrations (MICs). The time-kill test and ATP bioluminescence assay are introduced for their rapid and quantitative results, while flow cytometry is noted for its ability to detect antimicrobial resistance and cell damage but is limited by the availability of specialized equipment. The paper emphasizes the importance of standardized protocols and methodological adaptations when testing natural products to ensure accurate and reproducible results.