In vivo cross-linking mass spectrometry (XL-MS) enables proteome-wide characterization of protein complexes in live cells. However, current XL-MS methods face significant sample loss during enrichment, limiting their application to small sample quantities, and suffer from low reproducibility (20%-40%), hindering precise quantification. To address these challenges, we developed a novel membrane-permeable cross-linker, 2,6-dimethylpiperidine disuccinimidyl tridecanoate (DPST). The dimethylpiperidinyl group in DPST enables efficient cross-linked peptide enrichment via a one-step TMT antibody approach, eliminating sample loss from multi-step processes and allowing analysis with as few as 1E4 cells. Furthermore, DPST facilitates isotopic labeling (DPST-H/L), enabling light and heavy labeling of cross-linked samples at the cellular level, which reduces inaccuracies from multi-step preparations. This generates reporter ions for precise MS2 quantification, improving the signal-to-noise ratio without increasing spectral complexity. Using DPST (H/L), we successfully analyzed cross-links in primary neurons from single fetal mice and quantified transient and weak interactions, such as between hnRNPA2B1 and MKI67 in dynamic LLPS environments, further validated by biological analysis. Additionally, DPST’s design supports multiple isotopic labeling configurations (e.g., 6-plex, 10-plex). Therefore, our approach provides a powerful tool for both qualitative and quantitative XL-MS analysis of living systems, advancing proteomics and biomedical research even with limited sample quantities.