A simplified method for identifying aerobic actinomycetes using thin-layer chromatography (TLC) is described. The method involves analyzing whole cells for diaminopimelic acid (DAP) and carbohydrates, as well as the organism's ability to decompose casein, tyrosine, and xanthine. The TLC technique simplifies the process, making it suitable for routine clinical laboratories. The system was tested on 18 reference cultures and 35 clinical isolates, correctly identifying most of them. The method relies on the chemical composition of cell walls, including DAP isomers and carbohydrates, which are analyzed by TLC. DAP isomers are separated using a specific solvent system, while carbohydrates are identified using another solvent system. The results showed that the method is effective in identifying aerobic actinomycetes, including species such as Nocardia, Streptomyces, and Actinomadura. The method is efficient, requiring less time and resources than traditional paper chromatography. It is particularly useful in clinical settings for rapid identification and diagnosis of infections caused by these organisms. The study also highlights the importance of chemotaxonomic markers in the classification of microorganisms. The method has been successfully applied to identify various strains, including two isolates of Actinomadura dassonvillei. The system is reliable and practical for routine use in clinical microbiology laboratories.A simplified method for identifying aerobic actinomycetes using thin-layer chromatography (TLC) is described. The method involves analyzing whole cells for diaminopimelic acid (DAP) and carbohydrates, as well as the organism's ability to decompose casein, tyrosine, and xanthine. The TLC technique simplifies the process, making it suitable for routine clinical laboratories. The system was tested on 18 reference cultures and 35 clinical isolates, correctly identifying most of them. The method relies on the chemical composition of cell walls, including DAP isomers and carbohydrates, which are analyzed by TLC. DAP isomers are separated using a specific solvent system, while carbohydrates are identified using another solvent system. The results showed that the method is effective in identifying aerobic actinomycetes, including species such as Nocardia, Streptomyces, and Actinomadura. The method is efficient, requiring less time and resources than traditional paper chromatography. It is particularly useful in clinical settings for rapid identification and diagnosis of infections caused by these organisms. The study also highlights the importance of chemotaxonomic markers in the classification of microorganisms. The method has been successfully applied to identify various strains, including two isolates of Actinomadura dassonvillei. The system is reliable and practical for routine use in clinical microbiology laboratories.