Peningkatan kadar kolesterol dalam darah umumnya disebabkan oleh gangguan metabolisme lemak. Berdasarkan data Survei Kesehatan Indonesia tahun 2023, sebanyak 11,7% penduduk mempunyai kadar kolesterol tinggi. Meskipun terapi farmakologis dengan obat penurun kolesterol banyak digunakan, pemakaian dalam dosis tinggi dan jangka panjang dapat menimbulkan efek samping, seperti miopati. Oleh karena itu, diperlukan alternatif pengobatan yang lebih aman, salah satunya adalah penggunaan daun pepaya (Carica papaya), yang diketahui memiliki potensi dalam menurunkan kadar kolesterol. Penelitian ini bertujuan untuk mengevaluasi aktivitas fraksi etil asetat daun pepaya terhadap penurunan kadar kolesterol pada tikus Wistar yang diinduksi pakan tinggi lemak. Ekstraksi dilakukan dengan metode maserasi menggunakan etanol 70% selama 30 jam, diikuti proses fraksinasi dengan pelarut etil asetat. Pengukuran kadar kolesterol total, LDL, dan HDL dilakukan menggunakan spektrofotometer ABX Pentra. Analisis data mencakup uji normalitas Shapiro-Wilk, homogenitas Levene, uji paired sample t-test, dan One Way ANOVA. Hasil menunjukkan bahwa fraksi etil asetat secara signifikan menurunkan kadar kolesterol total, terutama pada dosis 80 mg/200 gr BB tikus, namun tidak memberikan pengaruh signifikan terhadap kadar LDL maupun HDL. / The increase in blood cholesterol levels is generally caused by lipid metabolism disorders. According to data from the 2023 Indonesian Health Survey, 11.7% of the population has high cholesterol levels. Although pharmacological therapy using cholesterol-lowering drugs is widely used, high doses and long-term use can lead to side effects such as myopathy. Therefore, safer alternative treatments are needed, one of which is the use of papaya leaves (Carica papaya), which are known to have potential in lowering cholesterol levels. This study aims to evaluate the activity of the ethyl acetate fraction of papaya leaves in reducing cholesterol levels in Wistar rats induced with a high-fat diet. Extraction was carried out by maceration using 70% ethanol for 30 hours, followed by fractionation with ethyl acetate solvent. Measurements of total cholesterol, LDL, and HDL levels were performed using the ABX Pentra spectrophotometer. Data analysis included the Shapiro-Wilk normality test, Levene’s homogeneity test, paired sample t-test, and One Way ANOVA. The results showed that the ethyl acetate fraction significantly reduced total cholesterol levels, especially at a dose of 80 mg/200 g body weight of rats, but did not have a significant effect on LDL or HDL levels.