During biotherapeutics cell line selection and process development, cell culture media provides essential nutrients for maintaining cell health and maximizing biotherapeutics production. The spent media is a complex mixture, containing hundreds of compounds from the starting media & feeds and metabolites generated during growth. The spent media assay is used to monitor daily changes of amino acids and metabolites. It is desirable to monitor metabolite accumulated since some are known to have a detrimental effect on product quality and titer. In this investigation, a Semi-Targeted HRMS based method was developed for routine media monitoring. The method consists of a library containing 200+ compound, a stepwise guided workflow for ease of data review, investigation tools, and a statistical, MVA tools for batch comparisons. Post data acquisition, cell culture media compound and potential metabolites were searched against a library containing 200+ compounds, containing retention time and fragmentation information based on pure standards. The data review process was simplified by a custom built stepwise guided workflow to review by compound class. Trend plots for single (or multiple) compounds plotted against all injections provided an immediate visualization of variation of the time courses and clones being analyzed. HRMS data on all components in the mixture allows preliminary identity elucidation of unknowns through Chem-spider database searching. In cell line development, this assay is used primarily to verify sequence variant misincorporation and identify metabolites & amino acids linked to critical product quality attributes. The results from this newly developed workflow are presented in this poster.