The use of biodegradable nanoparticles (NPs) as a carrier for therapeutic and diagnostic agents has gained immense attention in recent years worldwide. Due to their nano-size and high drug loading capacity they have several other characteristics over other nanoparticles including their role in biocompatibility and convenient release of the drug or any cargo agent at the target tumour site, these biological nanoparticles are used in a variety of applications. Due to the prolonged circulation time; erythrocytes or red blood cells (RBC's) can be used as potential drug delivery systems with the enhanced cargo-loading ability with less toxicity. In the present work, erythrocyte-derived nanovesicles were extracted from mice whole blood with consistent reproducibility. These nanovesicles, known for being efficient drug nanocarriers were encapsulated with and doxorubicin anticancer drug (1 mg/mL) for targeted cancer chemotherapy. To further understand the surface markers, present on these biologically derived nanovesicles we have used a mass spectrometry-based approach for identifying significant proteins and surface membrane lipids and their potential role as targeting ligands for cancer cells. Uniform particle size distribution is confirmed through dynamic light scattering measurements. Further cell viability and confocal microscopy studies showed the effective killing of cancer cells with these drug-loaded biocompatible nanocarriers.