Abstract: Off-gel fractionation and ¹⁸O labeling are widely used in proteomics research. In this study, two methods were applied in tryptic peptides from rat striatum protein. Results show that off-gel has better separation and isoelectric focusing for complex biological samples. Furthermore, ¹⁸O labeled peptides have no apparent back-exchange, and over 90% proteins from equal amounts of ¹⁶O and ¹⁸O labeled peptides mixture have a ¹⁶O/¹⁸O ratio of 1.04±0.16 after ultrafiltration, indicating no apparent effect on quantification and ¹⁸O labeling is compatible with off-gel separation based on ultrafiltration, which can provide an alternative separation method for ¹⁸O labeling and play important role in quantitative proteomics research.