Plasma is the most commonly employed matrix for analyzing fatty acids (FAs), but its extraction is not well accepted in the infant population. The objectives of this study were to evaluate cheek cells and capillary blood as alternatives to plasma sampling for FA analysis and to standardize the methodology. Samples were obtained from 20 children who underwent lipid extraction, phospholipid isolation by Solid Phase Extraction (SPE) in a 96-well plate, methylation, and analysis by fast gas chromatography (GC). A positive correlation was found for most of the FAs, especially long-chain polyunsaturated fatty acids (LC-PUFAs), in cheek cells and capillary blood versus plasma samples (r = 0.32-0.99). No differences were found in the levels of n-6: n-3 PUFA and n-6: n-3 LC-PUFA ratios between cheek cells and capillary blood. These two proposed samples can therefore be used as alternatives to plasma sampling for phospholipid FA analysis, especially LC-PUFAs.