Mass spectrometry (MS)-based targeted lipidomics enables the robust quantification of selected lipids under various biological conditions but comprehensive software tools to support such analyses are lacking. Here we present LipidCreator (LC), a software that fully supports targeted lipidomics assay development. LC offers a comprehensive framework to compute MS/MS fragment masses for over 60 lipid classes. LC provides all functionalities needed to define fragments, manage stable isotope labeling, optimize collision energy and generate in silico spectral libraries. We validate LC assays computationally and analytically and prove that it is capable to generate large targeted experiments to analyze blood and to dissect lipid-signaling pathways such as in human platelets.