The triacylglycerol (TAG) to sterol (ST) ratio, which has been used to determine the condition index in marine species, has regularly been measured via thin-layer chromatography with flame ionization detection (TLC/FID) using the Chromarod-Iatroscan system. However, this method is labor intensive, requires long analysis times, and has a detection limit of similar to 50 ng. For the determination of lipids in very small samples such as individual fish larvae, short-column gas chromatography with flame ionization detection (GC/FID) provides an excellent alternative to Iatroscan TLC/FID, owing to its lower detection limit (similar to 1 ng for high-molecular-weight TAG and similar to 0.1 ng for lower-molecular-weight species). As well, GC/FID individually profiles lipids based on their carbon number, whereas TLC/FID groups lipids according to their lipid class. Here we describe a method for the determination of neutral lipids from individual cyclopterus lumpus (lumpfish) and Myoxocephalus scorpius (shorthorn sculpin) larvae using short-column GC/FID. By using an internal standard and applying weight correction factors, ST and TAG can be accurately and precisely measured. The results reveal that there were no significant differences between GC/FID and TLC/FID in the quantification of TAG or ST in individual fish larvae (P > 0.05); however, GC/FID is more sensitive, precise, rapid, and cost-efficient.