<b>Objectives:</b> Uterine leiomyoma (ULM) variants, such as smooth muscle tumors of uncertain malignant potential (STUMP), may represent pre-malignant tumors that can lead to the development of malignant neoplasms, such as uterine leiomyosarcomas (LMS). Here we performed in-depth global proteomic analyses of archival ULM, STUMP, and LMS tissues from >60 patients to explore the relationship of protein expression among these tumor types. <b>Methods:</b> Diagnosis-confirmed, formalin-fixed, paraffin-embedded tissues corresponding to ULM (<i>n</i>=25), STUMP (<i>n</i>=16), and LMS (<i>n</i>=27) were obtained from the Joint Pathology Center. Whole tumor collections employing laser microdissection were trypsin digested and analyzed by multiplexed, mass spectrometry-based quantitative proteomics. Differential expression analysis was performed using commercial and in-house bioinformatic pipelines. <b>Results:</b> Global proteomic analysis of ULM, STUMP, and LMS tumor tissues quantified 7,090 unique proteins. Principle component analysis of 187 variably abundant proteins (median absolute deviation > 1) accounted for 32.6% and 12.6% of the variance observed between these tumor groups. Differential analysis of STUMP, LMS, and ULM tumors revealed 30 proteins significantly co-altered across samples (LIMMA adjusted p < 0.05). Many of these proteins exhibited progressively increasing abundance trends across ULM to STUMP to LMS tumors and correlated with enrichment of DNA replication and cell proliferation signaling pathways. Comparison of proteins co-altered in LMS versus STUMP and LMS versus ULM tumors further revealed enrichment of cellular pathways regulating cell proliferation as well as vasculogenesis signaling in LMS tumors. Additional investigation of proteins uniquely elevated in LMS tumors identified several candidates involved in regulating cellular metabolism and are targetable by small molecule inhibitors. <b>Conclusions:</b> Quantitative proteomic analysis of uterine smooth muscle tumors reveals distinct changes in the proteomes of ULM, STUMP, and LMS tumors, including increased tumor cell proliferation signaling in LMS compared with ULM. These data also identify discrete alterations correlating with the transition of ULM to STUMP to LMS tumors that may contribute to the molecular evolution from ULM to STUMP tumors and drive LMS to be aggressive, treatment-resistant, and deadly. The comparative analysis further revealed insights into enriched cellular pathways, and protein alterations uniquely elevated in LMS tumors, further correlating with vasculogenesis signaling and cellular metabolism that are putative therapeutic targets. This study provides insights into the disease biology underlying the development of ULM, STUMP, and LMS tumors that may lead to the development of diagnostic and treatment strategies toward improved management of these increasingly prevalent gynecologic conditions.Fig. 1