This review covers the state of the art of the analysis of real (or non-ideal) samples on microfluidic devices. A real sample analysis performed on microfluidics conceptually involves the complete integration of sample preparation, analyte separation, and detection on these platforms. Different "lab-on-a-chip" approaches have emerged in relevant application areas such as clinical, environmental, and food analysis which will be critically illustrated and discussed with respect to the strengths and weakness found. Likewise, the main challenges and perspectives will also be commented on.