Fatty acids are a well-established class of compounds targeted as biosignatures for future missions to look for evidence of life on ocean worlds such as Europa and Enceladus. In order to establish their abiotic or biotic origin, we need to separate and quantify fatty acids to determine their relative abundances within a sample. In this study, we demonstrate the high potential of capillary electrophoresis coupled to mass spectrometry (CE-MS) for the efficient separation and sensitive detection of a wide variety of fatty acids. Three derivatization strategies were evaluated to allow the detection of fatty acids by positive ionization mode MS. Furthermore, CE-MS conditions were optimized to provide maximum separation efficiencies and detection sensitivities for the analysis of saturated and unsaturated fatty acids with even- and odd-numbered carbon chain lengths. Optimum separation and detection were obtained using a background electrolyte of 2 M acetic acid in 45% acetonitrile, after derivatization of the fatty acids with 2-picolylamine or N,N-diethylethylenediamine. The limits of detection for the derivatized fatty acids using the optimized method ranged from 25 to 250 nM. The optimized method was also used for the analysis of fatty acids in cell cultures and natural samples. Two distinctive biosignatures were obtained for the microorganisms Halobacillus halophilus and Pseudoalteromonas haloplanktis. In addition, multiple fatty acids were detected in a natural sample from Mono Lake, California.