A crucial part of optimization of metabolically engineered organisms is producing balanced levels of pathway proteins. Typically, protein levels are monitored by Western blot analysis; however, application to multiple enzyme pathways can be difficult without unique antibodies for each enzyme in the pathway. Furthermore, it can be time consuming, and cost prohibitive during exploratory stages of pathway design when many different proteins must be monitored simultaneously. We present here a targeted proteomics approach that uses selected-reaction monitoring (SRM) mass spectrometry to quantify multiple proteins in a sample. SRM methods provide high selectivity and high sensitivity to enable rapid quantification of multiple proteins in an engineered pathway regardless of sequence or organism of origin.