Thin layer chromatography (TLC) is an efficient, fast and simple method of metal ion analysis that can be easily applied in the analysis of samples of various types of materials. Today, this method has a huge application in the analysis of historical and contemporary textile materials and is used to identify and quantify individual components in a sample. In historical samples on painted textiles, the TLC method is most often used in the analysis of waxes, resins, proteins and sugars, i.e. natural binders that are often found on historical samples. Unlike those materials, in modern materials it is possible to detect a wide range of other types of materials in coatings, which define special usage properties in the samples. Such materials are for example antimicrobially active biodegradable polymers that contain nanoparticles of metals and metal oxides present in the outer layer. Biodegradable polymers that have antimicrobial properties have a very wide application in medicine and medical materials and are often used as medical textiles (bedding and hospital towels) or consumables such as bandages, gauze, and wound dressings. Polymers that are not produced by fiber production and spinning technology, but by other processes (such as additive technology or 3D printing) can also be functionalized with nanoparticle coatings to obtain antimicrobial properties. These materials can therefore be used as catheters, consumables, coatings and bags. In this work, the TLC method was applied to analyze 64 different metal ions in the antimicrobial coating of samples prepared in the laboratory. The stationary phases tested were 31 different preprepared cellulose and silica gel, polyamid and Al2O3, and the mobile phase consisted of various developers containing mixtures of inorganic and organic solvents (such as acetonitrile, acetyl acetone, butanol, triethylamine, ethylenediaminotetraacetic acid, hydrochloric acid, water, etc.). During the experiment, 19 different solvent mixtures were used. The optimal system that detected all metal ions consisted of silica gel as a stationary phase, while the mobile phase contained a solvent mixture of acetonitrile: hydrochloric acid: water, a volume of 24 mL in a volume ratio of 60.00: 19.17: 20.83. Sample detection was performed using a UV cabinet and during the detection the separation parameters (RF, Rs and others) of known standard solutions of metal ions were determined, which were then used in the detection of analytes in an unknown samples. The new method was applied in the analysis of aqueous solutions of samples that had metal ion content below the detection limits, and therefore, before quantification, a method of adding a known standard metal content to unknown samples was performed. In this way, iron, chromium and cobalt were detected in three different real samples with very high repeatability and accuracy of the analysis.