Free or non-esterified fatty acids are the product of lipolysis of storage fat, i.e. triacylglyceroles. When the amount of fat exceeds the capacity of lipid-storing organs, free fatty acids affect and damage other non-lipid-storing organs. This process is termed lipotoxicity. Within a cell, free fatty acids can damage mitochondria, and lipotoxicity-induced mitochondrial damage has been associated recently with Peroxisomal Biogenesis Disorders. Drosophila melanogaster has a rising popularity as a model organism for metabolic diseases, but an optimized assay for measuring free fatty acids in Drosophila tissue samples is missing. Here we present a detailed protocol highlighting technical requirements and pitfalls to determine free fatty acids in samples of Drosophila tissue. The colorimetric assay allows the reproducible and cost-efficient measurement of free fatty acids in a 96 well plate format. We used our assay to determine changes in free fatty acid levels in different developmental stages and feeding conditions, and found that larvae and adults have different patterns of free fatty acid formation during starvation. Our assay is a valuable tool in the modeling of metabolic diseases with Drosophila melanogaster.