Thin-layer chromatography is a quick, simple and economic analytical method for separating different analytes. Hence the purpose of this work was to validate thin-layer chromatography as an analytical procedure allowing simultaneous determination of the benzodiazepines alprazolam, diazepam, oxazepam and the opioid methadone. Validation of testing quality was performed using two TLC systems: TA system involving methanol and ammonium mixed in a 100:1.5 ratio and Te system with dichloromethane and methanol mixed in a 9:1 ratio. In the process of validating the testing quality, selectivity needs to be tested and limit of detection must be set for each analyte. For selectivity testing, standard analyte solutions and certain auxiliary substances were prepared (matrix), so we could monitor the influence of the matrix on analyte separation. Limit of detection was established by preparing a set of dilutions of basic standard solutions; after chromatogram visualisation, the lowest concentration for detecting an analyte was set. Limits of detection are: alprazolam 0.80 μg/mL for TA system and 0.53 μg/mL for Te system; diazepam 3.14 μg/mL for TA and Te systems; oxazepam 3.53 μg/mL for TA system and 5.30 μg/mL for Te system, and methadone 6.09 μg/mL for TA system and 9 .14 μg/mL for Te system. Based on the validation results, we were able to confirm that TLC systems TA and Te are appropriate for identification of benzodiazepines like alprazolam, diazepam, oxazepam and of methadone and that auxiliary substances have no effect on the identification process.