Researchers are increasingly thinking smaller to solve some of the biggest challenges in nanomedicine: the control of drug encapsulation. Although recent years have witnessed a significant increase in the development and characterization of polymeric drug nanocarriers, several key features are still to be addressed: Where is the drug located within each nanoparticle (NP)? How much drug does each NP contain? Is the drug loading homogeneous on an individual NP basis? To answer these questions, individual NP characterization was achieved here by using atomic force microscopy-infrared spectroscopy (AFM-IR). A label-free quantification methodology was proposed to estimate with a nanoscale resolution the drug loadings of individual poly(lactic acid) (PLA) NPs loaded with an anticancer drug. First, a drug loading calibration curve was established using conventional IR microspectroscopy employing PLA/drug homogeneous films of well-known compositions. Then, single NPs were investigated by AFM-IR acquiring both IR mappings of PLA and drug as well as local IR spectra. Besides, drug location within single NPs was unravelled. The measured drug loadings were drastically different (0 to 21 wt%) from one NP to another, emphasizing the particular interest of this methodology in providing a simple quantification method for the quality control of nanomedicines.