The control mechanisms of de novo fatty acid biosynthesis continue to intrigue, particularly at protein level. The cross talk between transcriptional and metabolic regulatory networks remains undefined. In order to understand the metabolic factors affecting oleic acid biosynthesis in Elaeis guineensis var. tenera (oil palm), two-dimensional liquid chromatography-tandem mass spectrometry (2DLC-MS/MS) was attempted to capture the main fatty acid biosynthetic enzymes involved in oleic acid production. These enzymes were extracted from the chromoplast of low and high oleic mesocarps of 20th week after anthesis. Chromoplast proteins were separated using offline 2DLC for mass spectrometric analysis. Two subunits of acetyl-CoA carboxylase, β-ketoacyl-acyl carrier protein (ACP) reductase, β-hydroxyacyl-ACP dehydrogenase, 3-enoyl- ACP reductase, β-ketoacyl-ACP synthase and stearoyl-ACP desaturase were identified. The relative expression of these fatty acid biosynthetic enzymes was determined using isobaric tags labelling. Initial assessment revealed that β-hydroxyacyl-ACP dehydrogenase and β-ketoacyl-ACP synthase were present in a higher abundance in the high oleic acid mesocarps while the accumulation of 3-enoyl-ACP reductase was lower relative to the other fatty acid biosynthetic enzymes investigated. The existence of other differentially expressed metabolic enzymes suggests that the control mechanism of fatty acid production, in particular oleic acid, might involve more than just the main fatty acid biosynthetic enzymes.