Uterine cancers are among the most prevalent gynecological malignancies, and endometrial cancer (EC) is the most common in this group. This study used tissue-based proteomic profiling analysis in patients with endometrial cancer and hyperplasia, and control patients. Conventional 2D gel electrophoresis, followed by a mass spectrometry approach with bioinformatics, including a network pathway analysis pipeline, was used to identify differentially expressed proteins and associated metabolic pathways between the study groups. Thirty-six patients (twelve with endometrial cancer, twelve with hyperplasia, and twelve controls) were enrolled in this study. The mean age of the participants was 46-75 years. Eighty-seven proteins were significantly differentially expressed between the study groups, of which fifty-three were significantly differentially regulated (twenty-eight upregulated and twenty-five downregulated) in the tissue samples of EC patients compared to the control (Ctrl). Furthermore, 26 proteins were significantly dysregulated (8 upregulated and 18 downregulated) in tissue samples of hyperplasia (HY) patients compared to Ctrl. Thirty-two proteins (nineteen upregulated and thirteen downregulated) including desmin, peptidyl prolyl cis-trans isomerase A, and zinc finger protein 844 were downregulated in the EC group compared to the HY group. Additionally, fructose bisphosphate aldolase A, alpha enolase, and keratin type 1 cytoskeletal 10 were upregulated in the EC group compared to those in the HY group. The proteins identified in this study were known to regulate cellular processes (36%), followed by biological regulation (16%). Ingenuity pathway analysis found that proteins that are differentially expressed between EC and HY are linked to AKT, ACTA2, and other signaling pathways. The panels of protein markers identified in this study could be used as potential biomarkers for distinguishing between EC and HY and early diagnosis and progression of EC from hyperplasia and normal patients.