Despite continuous technological improvements in sample preparation, mass-spectrometry-based proteomics for trace samples faces the challenges of sensitivity, quantification accuracy and reproducibility. Herein, we explored the applicability of turboDDA (a method by using data-dependent acquisition without dynamic exclusion) for quantitative proteomics of trace samples. After systematic optimization of acquisition parameters, we compared the performance of turboDDA with data-dependent acquisition with dynamic exclusion (DEDDA). By benchmarking the analysis of trace unlabeled human cell digests, turboDDA showed substantially better sensitivity in comparison with DEDDA, no matter for unfractionated or high pH fractionated samples. Furthermore, through designing an iTRAQ labeled three-proteome model (i.e., tryptic digest of protein lysates from yeast, human and E. coli) to document the interference effect, we evaluated the quantification interference, accuracy, reproducibility of iTRAQ labeled trace samples, and the impact of PIF (precursor intensity fraction) cutoff for different approaches (turboDDA and DEDDA). The results showed that improved quantification accuracy and reproducibility could be achieved by turboDDA while more stringent PIF cutoff resulted in more accurate quantification but less peptide identification for both approaches. Finally, the turboDDA strategy was applied to the differential analysis of limited amounts of human lung cancer cell samples, showing great promise in the trace proteomics sample analysis.