We have developed an approach to perform "on the fly" electron spin resonance (OTF-ESR) measurements of negative bias temperature instability (NBTI) defect generation. This OTF-ESR approach allows for an atomic-scale identification of the defects involved in NBTI free of any recovery contamination. We demonstrate that, during NBTI stressing at elevated temperature and modest negative oxide bias, positively charged oxygen vacancy sites (E′ centers) are generated. Upon removal of the NBTI stressing conditions, the E′ center density quickly recovers to that of its pre-stress values. When similar measurements are made with zero oxide bias at elevated temperature or negative oxide bias at room temperature, the E′ defect density does not change. These observations strongly indicate that NBTI is triggered by inversion layer hole capture at an E′ precursor site which then leads to the depassivation of nearby interface states.