PCR-based methods have caused a surge in the integration of eco-physiological approaches into research on partial nitritation anammox (PNA). PNA systems have been characterized as fine-tuned biological nitrogen removal (BNR) process with a very complex ecosystem. Therefore, molecular methods, which offer a wide range of approaches comparable to a workman’s toolbox, have been intensively used to understand these PNA systems and achieve a stable process. On the one hand, quantitative PCR (qPCR) became the most com-mon method to quantify target microorganisms in engineered systems such as PNA and oth-er ecological studies and is therefore the so-called gold standard for a fast and reliable quantification. On the other hand, next-generation sequencing (NGS) as a new and ad-vanced approach enabled in-depth analysis, provided new genomes in public databases, and resulted in a more conscious look on the PNA microbiome. All research work combined in this study revealed a framework to overcome challenges for better integration of the molecular methods in wastewater microbiome studies - which is mainly about understanding the current biases in molecular methods, standardization, and selection of the right combination of molecular methods. In general, data consistency and accuracy strongly depend on the primer selection and data interpretation. The reevaluation of existing primers and the design of a more specific primer will improve the respective mo-lecular studies and support our understanding, which then leads to an improved assessment of nitrification-denitrification (N-DN) and PNA systems. The combination of traditional microbiology and the modern molecular biological methods has received only marginal attention in this work but will be the non-plus ultra-method for further insights into complex microbiomes.