Aconiti Lateralis Radix Praeparata (Fuzi) is a commonly used traditional Chinese medicine in clinic for its potency in restoring yang and rescuing from collapse. Aconiti alkaloids, mainly including monoester-diterpenoidaconitines (MDAs) and diester-diterpenoidaconitines (DDAs), are considered to act as both bioactive and toxic constituents. In the present study, a feasible, economical, and accurate HPLC method for simultaneous determination of six alkaloid markers using the Single Standard for Determination of Multi-Components (SSDMC) method was developed and fully validated. Benzoylmesaconine was used as the unique reference standard. This method was proven as accurate (recovery varying between 97.5%–101.8%, RSD < 3%), precise (RSD 0.63%–2.05%), and linear (R = 0.999 9) over the concentration ranges, and subsequently applied to quantitative evaluation of 62 batches of samples, among which 45 batches were from good manufacturing practice (GMP) facilities and 17 batches from the drug market. The contents were then analyzed by principal component analysis (PCA) and homogeneity test. The present study provided valuable information for improving the quality standard of Aconiti Lateralis Radix Praeparata. The developed method also has the potential in analysis of other Aconitum species, such as Aconitum carmichaelii (prepared parent root) and Aconitum kusnezoffii (prepared root).