By integrating LipidCreator for targeted lipid library generation and PRM Conductor for streamlined parallel reaction monitoring (PRM) method setup, Skyline simplifies method development while ensuring efficient and reproducible data acquisition. Its intuitive data analysis tools further refine lipid quantification, allowing users to confidently analyze complex lipid panels with minimal manual intervention. To demonstrate this, we applied a Skyline-driven workflow to quantify over 1000 lipids in NIST 1950 plasma within a 12-minute acquisition on the Stellar MS. The integration of LipidCreator enabled the construction of an enhanced targeted list, incorporating multiple transitions per lipid species. This resulted in an average of 3.5 transitions per analyte, increasing lipid species diversity by 50% while improving peak definition and scan density by at least 20%. This work highlights Skyline’s role in advancing lipidomics, providing researchers with a flexible, scalable, and user-friendly solution for high-confidence lipid panel development. By leveraging automated method generation, streamlined PRM acquisition, and detailed data analysis, Skyline enables more efficient lipidomics research, enhancing quantitative reliability, specificity, and throughput for emerging mass spectrometry technologies.