The quantification of free cholesterol (FC) and cholesteryl ester (CE) in mammalian samples is of great interest for basic science and clinical lipidomics. Here, we evaluated the feasibility of direct flow injection analysis (FIA) coupled to electrospray ionization high-resolution mass spectrometry (ESI-HRMS) to quantify FC and CE in lipid extracts from human serum, cultured cells, and mouse liver. Despite poor ionization efficiency of FC, the limit of quantitation was sufficient for precise and accurate quantification of FC by multiplexed HRMS (MSX) analysis without using a derivatization step. However, it was demonstrated that, upon full scan Fourier transform MS (FTMS) quantification, CE species show substantial differences in their analytical responses depending on number of double bonds, length of the acyl chain, infused lipid concentration, and other lipid components. A major determinant for these response differences is their susceptibility to in-source fragmentation. In particular, introduction of double bonds lowers the degree of in-source fragmentation. Therefore, CE species-specific response factors need to be applied for CE quantification by FTMS to achieve accurate concentrations. Method validation demonstrated that FIA-ESI-HRMS (MSX and FTMS) is applicable for quantification of FC and CE in samples used in basic science as well as clinical studies such as cultured cells, tissue homogenates, and serum.