PubChem serves as a comprehensive repository, housing over 100 million unique chemical structures representing the breadth of our chemical knowledge across numerous fields including metabolism, pharmaceuticals, toxicology, cosmetics, agriculture, and many more. Rapid identification of these small molecules increasingly relies on electrospray ionization (ESI) paired with tandem mass spectrometry (MS/MS), particularly by comparison to genuine standard MS/MS data sets. Despite its widespread application, achieving consistency in MS/MS data across various analytical platforms remains an unaddressed concern. This study evaluated MS/MS data derived from one hundred molecular standards utilizing instruments from five manufacturers, inclusive of quadrupole time-of-flight (QTOF) and quadrupole orbitrap "exactive" (QE) mass spectrometers by Agilent (QTOF), Bruker (QTOF), SCIEX (QTOF), Waters (QTOF), and Thermo QE. We assessed fragment ion variations at multiple collisional energies (0, 10, 20, and 40 eV) using the cosine scoring algorithm for comparisons and the number of fragments observed. A parallel visual analysis of the MS/MS spectra across instruments was conducted, consistent with a standard procedure that is used to circumvent the still prevalent issue of mischaracterizations as shown for dimethyl sphingosine and C20 sphingosine. Our analysis revealed a notable consistency in MS/MS data and identifications, with fragment ions' m/z values exhibiting the highest concordance between instrument platforms at 20 eV, the other collisional energies (0, 10, and 40 eV) were significantly lower. While moving toward a standardized ESI MS/MS protocol is required for dependable molecular characterization, our results also underscore the continued importance of corroborating MS/MS data against standards to ensure accurate identifications. Our findings suggest that ESI MS/MS manufacturers, akin to the established norms for gas chromatography mass spectrometry instruments, should standardize the collision energy at 20 eV across different instrument platforms.