High-performance thin-layer chromatography (HPTLC) and high-performance liquid chromatography (HPLC) were established for the simultaneous determination of two anthocyanins in medicine mulberry, a specialty medicinal herb of Xinjiang. For the HPTLC method, a silica gel GF254 plate was used with formic acid‒water‒n-butanol (9.1:8.4:32.5, V/V) as the unfolding system, and the dispensing volume was 2 µL, which was unfolded at room temperature, and then examined under visible light, and scanned at 550 nm for quantitative analysis. HPLC method was performed on a Hypersil ODS2 C18 column (4.6 mm × 250 mm, 5 µm) with isocratic elution of acetonitrile‒0.2% aqueous phosphoric acid (9:91, V/V) at a flow rate of 1.0 mL/min with an injection volume of 10 μL, column temperature of 30 °C, and the analytes were analyzed by an ultraviolet detector at 520 nm. Both developed methods were also validated for the specificity, linearity and range, limit of detection (LOD) and limit of quantification (LOQ), intra- and interday precision, recovery, and stability. Results showed that the HPTLC methods for cyanidin-3-O-glicoside (C3G) and cyanidin-3-O-rutinoside (C3R) showed good linearity (R2 = 0.9988 and R2 = 0.9983) in the range of 0.2‒1.0 μg/spot; LOD values of C3G and C3R were 0.048 μg/spot and 0.036 μg/spot, LOQ values were 0.15 μg/spot and 0.11 μg/spot; intra-day precision (percentage relative standard deviation; %RSD) values were 1.32 and 3.62, inter-day precision (%RSD) values were 2.67 and 3.04, respectively; recoveries for C3G and C3R were 93.33‒102.78% and 95.83‒103.3%; and the sample solutions were stable within 90 min. The HPLC method showed good linearity (R2 = 0.9991, R2 = 0.9993) in the ranges of 12.24‒42.84 μg/mL for C3G and 8.16‒28.56 μg/mL for C3R, respectively. LOD values of C3G and C3R were 1.27 µg/mL and 0.73 µg/mL and LOQ values were 3.81 µg/mL and 2.20 µg/mL, respectively. Intra-day precision (%RSD) values were 0.64 and 0.98, and inter-day precision (%RSD) values were 0.54 and 0.65, respectively. Recoveries of C3G and C3R were 94.57‒98.85% and 93.27‒101.4%, the mixed standard solution was stable within 24 h, and the sample solution was stable within 8 h. The results of methodological validation were in agreement with the 2020 edition of the Chinese Pharmacopoeia, and two methods can be used for the determination of the content of medicine mulberry anthocyanins.