Tandem mass spectrometry, also known as MS/MS or MS2, involves multiple steps of mass spectrometry selection, with some form of fragmentation occurring in between the stages. Mass spectrometry is a powerful technique for chemical analysis that is used to identify unknown compounds, to quantify known compounds, and to elucidate molecular structure. To understand the principle of operation it is established that a mass spectrometer is a “molecule smasher” that measures molecular and atomic masses of whole molecules, molecular fragments and atoms by generation and detection of the corresponding gas phase ions, separated according to their mass-to-charge ratio (m/z). It measures masses correspond to molecular structure and atomic composition of parent molecule and hence allows determination and elucidation of molecular structure [1]. Now the pertinent question comes to mind that why mass spectrometry? It may also be used for quantitation of molecular species. It is considered a very sensitive technique and works with minute quantities of samples (as low as 10−12 g, 10−15 mol) and is easily interfaced with chromatographic separation methods for identification of components in a mixture. Mass spectrometry also provides valuable information to a wide range of professionals: chemists, biologists, physicians, astronomers, environmental health specialists. It works by generating spectrum by separating ions of different mass to charge ratio (m/z) where m is the molecular or atomic mass, z is the electrostatic charge unit. In many cases (such as small molecules), z = 1 measured m/z = mass of fragment. But this is not always true for large bio-molecules analyzed by electro spray (ESI), z > 1 [2]. Tandem mass spectrometer is of many different types—each has different advantages, draw-backs and applications. All consist of four major sections linked together inlet–ionization source–analyser–detector. All sections are usually maintained under high vacuum and the functions of instrument control, sample acquisition and data processing are under computer control. Data system and computer control is often overlooked—most significant advance in mass spectrometry—allows 24/7 automation and development of modern powerful analytical techniques [3]. Tandem mass spectrometer is a single instrument using two (or more) mass analyzers. Simplest form consists of two mass spectrometers (MS/MS) in series connected by a chamber known as a collision cell. The sample to be examined is essentially sorted and weighed in the first mass spectrometer, then broken into pieces in the collision cell, and a piece or pieces sorted and weighed in the second mass spectrometer. The tandem mass spectrometer is constructed of two or more quadrupoles, with a collision cell separating each quadrupole. Once a sample has been separated by chromatography, the substances initially go through an initial quadrupole which separates the mixture of ions allotting only certain ions (precursor ions), passage to the collision cell. The first quadrupole is used to select user-specified sample ions from a specific component; usually the molecular-related [i.e. (M + H) + or (M − H)−] ions within the collision cell the precursor-ions also known as “parent ions” are then bombarded with an inert gas (Xe, Ar, etc.) and are further broken down into different charged and mass ions (product ions). These product ions also known as “daughter ions” are then run through an additional quadrupole to further separate the ions which is set to monitor specific ion fragments. This process can be repeated several times in order to get highly specific readings [4]. There are several applications of tandem mass spectrometer. Clinical testing and toxicology, inborn errors of metabolism—newborn screening, cancer, diabetes, various poisons, drugs of abuse, etc. Biotechnology and pharmaceutical to determine chemical structure of drugs and drug metabolites, detection/quantification of impurities, drugs and their metabolites in biological fluids and tissues. High through-put drug screening, analysis of liquid mixtures, fingerprinting, nutraceuticals/herbal drugs/tracing source of natural products or drugs and many more. Protein sequencing and identification protein Identification via database search (SPC and spectral alignment), de novo peptide sequencing (spectrum graph), hybrid, identifying post translationally modified (PTM) peptides quantitative proteomics, identifying proteins that are differentially abundant, besides it has a significant role in proteomics [5, 6].