Abstract: Scientists have identified a large amount of protein/peptide biomarker candidates based on high throughput proteomics research in recent years. In order to transform the novel biomarkers from research to clinical utility, the accurate determination of the amount of protein and peptides is essential. Traditional immunoassays like ELISA holds lots of restrictions with regarding to the difficulty to obtain antibodies of mutant proteins, the existence of cross-activity, the low specificity for some candidate proteins, and the discrepancy between production batches. Owning to the great development of LC-MS/MS, technologies based on LC-MS/MS have been widely applied in quantification research of proteins and metabolites. Targeted proteomics technology such as SRM/MRM, PRM and MRM3 combined with protein/peptide standards can offer the advantage of accurate quantitation of interesting proteins or peptides. Many MS-based strategies of absolute quantification for proteins/peptides spring up, including strategies based on peptide standards, intact proteins and labeling reagents. Here, the current progress and the challenge of LC-MS/MS based absolute quantification strategies were summarized in detail, and the advantages and limitations of various strategies were compared. The applications in biomedical research of these strategies based on LC-MS/MS were also listed. Of course, some recommendations, as peptide standards choosing, processing, storage, the construct of standard curve and strict control of the pipeline according to published research, were currently available surrounding the improvement of quantification accuracy, and the technical transformation from research to clinic. We hope these summaries and advice would be helpful for choosing proper strategy according to the research purpose and the accurate quantification of proteins or peptides.