IntroductionPrunellae Spica (PS), derived from the dried fruit spikes of Prunella vulgaris L., is a traditional Chinese medicinal herb. Our previous studies found that PVE30, a water‐extracting ethanol‐precipitating “glycoprotein” macromolecule of PS, was a potential anti‐herpes simplex virus (HSV) candidate. However, due to the complex structure and diverse bioactivity of the “glycoprotein”, ensuring its quality consistency across different batches of PVE30 becomes particularly challenging. This poses a significant hurdle for new drug development based on PVE30.ObjectiveOur study aimed to integrate multi‐index determination coupled with hierarchical cluster analysis (HCA) to holistically profile the quality consistency of “glycoprotein” in PVE30.MethodsHigh‐performance gel permeation chromatography with refractive index detector (HPGPC‐RID) was used to characterise the molecular weight (Mw) distribution, HPLC‐PDA was used to quantitatively analyse the composed monosaccharides and amino acids, and UV‐VIS was used to quantify the contents of polysaccharides and proteins. Qualitative and quantitative consistency was analysed for each single index in 16 batches of PVE30, and a 16 × 38 data matrix, coupled with HCA, was used to evaluate the holistic quality consistency of PVE30.ResultsThe newly developed and validated methods were exclusive, linear, precise, accurate, and stable enough to quantify multi‐indexes in PVE30. Single‐index analysis revealed that 16 batches of PVE30 were qualitatively consistent in Mw distribution, polysaccharides and proteins, and the composition of composed monosaccharides and amino acids but quantitatively inconsistent in the relative contents of some “glycoprotein” macromolecules, as well as the composed monosaccharides/amino acids. HCA showed that the holistic quality of PVE30 was inconsistent, the inconsistency was uncorrelated with the regions where PS was commercially collected, and the contents of 17 amino acids and 2 monosaccharides contributed most to the holistic quality inconsistency.ConclusionMulti‐index determination coupled with HCA was successful in evaluating the quality consistency of PVE30, and the significant difference in quantitative indices was not caused by the origin of PS. The cultivating basis should be confirmed for PVE30‐based new drug development.