A PCR-based method for detecting the absence of mycoplasmas (Mollicutes) was established in the laboryitories of the microbiological quality control of Novartis Vaccines & Diagnostics (NVD) in Marburg in this doctoral thesis. The objective was to create the bases for the subsequent validation and approval of this method by the authorities, so that both the test methods currently used for the release of vaccines (cultivation test and indicator cell test in accordance with the European Pharmacopoeia) can be replaced by the new procedure. To this end the most suitable method for the PCR-based detection of Mollicutes was the CytoInspectTM System made by Greiner Bio-One GmbH, the principle of which is based on a touchdown PCR, followed by a microarray analysis for the detection and identification of PCR products. Three ready-to-use kits were considered in direct comparison. The main criteria were the sensitivity measured in practical trials for samples with high cell (>107 cells/ml) and virus concentrations, the specificity, and the positive controls for monitoring the functionality of each individual test. Reference standards for ten different Mollicutes species were established for the validation of the PCR-based procedure and comparison with the previous cultivation-based methods. These were aliquoted and deep-frozen cell suspensions. The main criterion for the suitability of these standards was the lowest and most uniform ratio possible of gene copies (GC) per colony forming unit (CFU) in the individual aliquots of the respective cell suspensions. All ten reference standards exhibited a stable titer (CFU/ml) and a ratio of < 20 GC/CFU. The sensitivity of the CytoInspectTM Kit was determined in the form of the detection limit. For the ten Mollicutes species tested in this study this lay between 22 and 0.5 GC/ml (which corresponded to 4.5 – 0.1 CFU/ml), in a sample volume of 10 ml. It was comparable with the detection limit of the cultivation test, which was measured in this study at 2 to 0.1 CFU/ml. Due to the likewise high sensitivity in the detection of free DNA from cells which had already died this method also offers the possibility of preventing living contaminations in the production facilities. The robustness of the procedure and the specific (exclusive) detection of Mollicutes with the CytoInspectTM Kit could be demonstrated by the practical tests. The safety of the test procedure was assessed in the context of establishing the method as a routine application for the batch release of vaccines under the local conditions prevailing in the laboratories of the microbiological quality control of NVD in Marburg. This took place, on the one hand, through the evaluation of the critical steps which could lead to a false-negative result. On the other hand, the risk of false-positive results, which could occur due to contaminations of the samples during the conduction of the tests, was assessed and minimized through corresponding measures.