Protein lactylation is a novel post-translational modification (PTM) involved in many important physiological processes such as macrophage polarization, immune regulation, and tumor cell growth. However, traditional methodologies for studying lactylation have predominantly relied on peptide enrichment from whole-cell lysates, which tend to favor the detection of high-abundance peptides, thus limiting the identification of low-abundance lactylated peptides. To address this limitation, here, we employed subcellular fractionation to separate proteins and map lactylated peptides from each isolated subcellular fraction using a model cell line. In brief, we identified 1,217 lysine lactylation (Kla) sites on 553 proteins across four subcellular fractions. Subsequent pathway enrichment analysis revealed that Kla proteins participate in distinct pathways depending on the subcellular contexts. In addition, this subcellular fractionation method enabled the discovery of 36 previously unreported Kla proteins and 223 novel Kla sites, many of which are present in low abundance. Notably, several proteins contain multiple newly identified Kla sites, exemplified by the transcriptional regulator ATRX. Furthermore, our results indicate the possibility of PTM crosstalk between Kla and other PTMs such as ubiquitination and sumoylation. In conclusion, subcellular fractionation facilitates the identification of Kla proteins that have been previously uncovered and could be overlooked by affinity enrichment of whole-cell lysates.