Drug-containing nanoparticles, the foundation of nanomedicine, provide promise for the safe and effective delivery of drugs to their targets. In this study, we developed a simple method to determine the relative quantities of nanoparticle-encapsulated drugs by HPLC using a commercially available monolithic silica column. Amphotericin B- and irinotecan-containing nanoparticles produced nearly simultaneous elution peaks (~7 min), suggesting that elution was largely driven by hydrodynamic effects and was relatively unaffected by differences in the encapsulated drug. A good correlation was observed between the intensity of the nanoparticle peak and the relative quantity of encapsulated drug. We used our method to characterize the effects of drug quantity and nanoparticle size on drug encapsulation rates within the nanoparticles. Encapsulation increased with increasing quantities of the drug in the preparation solution. This effect was greater for irinotecan than for amphotericin B. Although absolute encapsulation also increased with increasing nanoparticle size, encapsulation efficiency decreased. Thus, the monolith column is suitable for evaluating nanomedicine quality and may be used to evaluate many kinds of nanomaterials. Graphical Abstract Evaluation method of quantity of drug encapsulated within nanoparticles was developed. The method can be applicable for a rapid quality assurance of nanomedicine.