Primycin is a macrolide antibiotic complex produced in microbiological fermentation processes. The microbial production of primycin requires an in-process analytical method suitable for monitoring the level of the active agents. In this paper, a method fulfilling the described requirement is presented. This method consists of a simple, efficacious extraction step, an instrumental sample application followed by a high-performance thin-layer chromatographic separation in relatively short time and a quantitative chromatogram evaluation. A dipping technique, in a solution containing sulfuric acid followed by heating at 120 degrees C, is used for chromogen formation, resulting in an absorption maximum at 290 nm. A progress diagram of the fermentation obtained by this technique is compared with one obtained by a microbiological agar diffusion method. The bioautographic evaluation of the active spots in the chromatogram are also presented. By our TLC method, the group of the active primycin components in the fermentation broth and by-products formed during the fermentation can be well separated. The relative intensities of the different TLC spots provide some information on the formation of the active components.