Mass spectrometry (MS) is a very powerful instrument that can be used to analyze a wide range of materials such as proteins, peptides, DNA, drugs, and polymers. The process typically involves either chemical or electron (impact) ionization of the analyte. The resulting charged species or fragment is subsequently identified by the detector. Usually, single mass uses source-induced dissociation (SID), whereas mass/mass uses collision-induced dissociation (CID) to analyze the chemical fragmentations Each technique has its own advantages and disadvantages. While CID is most effective for the analysis of pure substances, multiple-step MS is a powerful technique to get structural data. Analysis of veterinary drugs ampicillin, chloramphenicol, ciprofloxacin, and oxytetracycline serves to highlight the slight differences between SID and CID. For example, minor differences were observed between ciprofloxacin and oxytetracycline via SID or CID. However, distinct fragmentation patterns were observed for ampicllin depending on the analysis method. Both SID and CID showed similar fragmentation spectra but different signal intensities for chloramphenicol. There are several factors that can influence the fragmentation spectra, such as the collision energy, major precursor ion, electrospray mode (positive or negative), and sample homogeneity. Therefore, one must select a fragmentation method on an empirical and case-by-case basis.