Objective Currently, powdered Chinese herbal medicines(CHMs) were mainly evaluated through physical property, chemical dissolution, and bioactivity independently. It could not reflect the quality comprehensively. This paper was to explore and establish a comprehensive evaluation method for powdered CHMs. Methods Isatidis Radix was chosen as an exemple. Firstly, powdered Isatidis Radix in different particle size was prepared. Then, their physical properties were characterized. The dissolution of index component epigoitrin was determined, and their antiviral activities were evaluated by neuraminidase-based bioassay. Results As the particle size decreased, powder distribution tended to be uniform, and the dissolution of epigoitrin increased, antiviral activity enhanced. According to cluster analysis of above results, the sequence of evaluation consequence was ultrafine powder S2(D90: 32.80 ± 0.29) > ultrafine powder S1(D90: 52.08 ± 0.53) > fine powder S0(D90: 118.16 ± 0.76)(from the superior to the inferior). Conclusion Overall, the comprehensive evaluation for powdered CHMs based on the physical characterization, chemical dissolution, and bioassay could not only be used to evaluate powdered herbs, but also guide the screening and optimization of the particle size of powder. Objective Currently, powdered Chinese herbal medicines (CHMs) were mainly evaluated through physical property, chemical dissolution, and bioactivity independently. It could not reflect the quality comprehensively. This paper was to explore and establish a comprehensive evaluation method for powdered CHMs. Methods Isatidis Radix was chosen as an exemple. Firstly, powdered Isatidis Radix in different particle size was prepared. Then, their physical properties were characterized. The dissolution of index component epigoitrin was determined, and their antiviral activities were were evaluated by neuraminidase-based bioassay. Results As the According to cluster analysis of the above results, the sequence of evaluation was was ultrafine powder S2 (D90: 32.80 ± 0.29)> ultrafine powder S1 (D90: 52.08 ± 0.53)> fine powder S0 (D90: 118.16 ± 0.76) (from the su Perior to the inferior. Conclusion Overall, the comprehensive evaluation for powdered CHMs based on the physical characterization, chemical dissolution, and bioassay could not only be used to evaluate powdered herbs, but also guide the screening and optimization of the particle size of powder.