Nano-Drug Delivery Systems have emerged as an effective means of targeting therapeutic agents such as drugs, peptides and proteins to various target sites. The unique advantages and potential targeting capability of this approach have claimed it as the drug delivery system of the future. Various analytical methodologies have been used for the evaluation of this delivery system both in vitro and in vivo. This review will attempt to describe the various in vitro methods used for this purpose, their importance and limitations. The in vitro methods include particle sizing, zeta potential, microscopy, Fourier transform infrared (FTIR) spectroscopy, differential scanning calorimetry, X-ray diffraction, in vitro release characteristics, determination of drug loading, encapsulation efficiency and loading efficiency. In vitro cell culture has also been utilized in cellular uptake, transmission electron microscopy, MTT assays, flow cytometry, and cellular transport studies. In summary, this in-depth review will provide the basic understanding, as well as the pros and cons of commonly used analytical methods currently utilized to evaluate this emerging drug delivery system.