The high throughput characterization of protein N-termini is becoming an emerging challenge in the proteomics and proteogenomics fields. The present study describes the N-terminal oriented analysis of human mitochondria enriched samples by the trimethoxyphenyl phosphonium (TMPP) labeling approach (N-TOP and dN-TOP approaches (Gallien et al, 2009, Bertaccini et al. 2013)). The dN-TOP strategy based on a double light/heavy TMPP labeling has been optimized in order to improve and automate the workflow for efficient, fast and reliable high throughput N-terminome analysis.