Significant increases in the presence of organofluorine compounds, especially per- and polyfluoroalkyl substances (PFAS), have been reported in the various environmental compartments. Many of these compounds, including their precursors and metabolites, remain challenging to detect and quantify using current analytical methods. PFAS have the potential to undergo metabolic transformation processes under biotic conditions in the presence of suitable microbial communities and under varying environmental conditions, each with its unique metabolic pathways. Yet microbial degradation pathways remain poorly understood, particularly regarding the transformation of long-chain PFAS to shorter, presumably less toxic compounds. This study investigates the complete microbial transformation of perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS), two legacy PFAS compounds in the presence of different wastewater cultures and introduces a targeted analytical method for evaluating short-chain transformation products in complex matrices. The study employs biotic processes using microbial cultures, reducing the need for energy-intensive chemical or physical treatment methods. Aerobic cultures, including activated sludge and nitrifying sludge, demonstrated significant degradation of PFOA and PFOS after 21 days of exposure, with rapid decrease of the concentration observed after the third day.The identity and quantity of organofluorine products is evaluated using a combination of liquid chromatography tandem mass spectrometry (LC-MS/MS), liquid chromatography-high resolution mass spectrometry (LC-HRMS), ion-mobility spectrometry, and combustion ion chromatography (CIC) with particular emphasis on the applicability of each technique in evaluating PFAS removal, degradation and mass balance analyses. Impacts of biotic treatment on the PFAS was evaluated using via targeted LC-MS/MS analysis, including to selectively quantify shorter chain homologues as transformation byproducts. transformation byproducts of PFOA and PFOS, respectively. Suspect screening via liquid chromatography-high resolution mass spectrometry elucidated the presence of transformation byproducts not included in the targeted method. Ion mobility separation is used to separate the co-occurring isomers and enabled evaluation of changes in the respective isomer profiles during bacterial degradation. CIC following total extractable organofluorine analysis (EOF) enabled analysis of changes in the total organofluorine profile, which encompasses both native PFAS and possible transformed products during and after degradation experiments. Future work could enhance sustainability by further integrating green analytical techniques, minimizing solvent use during sample preparation, and focusing on scalable bioremediation strategies. These efforts aim to improve environmental and human health outcomes by promoting the degradation of persistent pollutants and reducing reliance on hazardous remediation methods.