A series of amino acid-based surfactants with a fixed alkyl chain length and with two carboxyl groups separated by a spacer of one, two or three carbon atoms have been synthesized and evaluated as potential collectors for flotation of calcite and fluorite. A monocarboxylate amino acid-based surfactant having the same length of the hydrocarbon tail was also included in the study. Experiments using a Hallimond flotation tube showed that although the flotation reagents solely differs in terms of spacer, their efficacy in terms of flotation recovery varied very much. Whereas on calcite at pH 10.5 only the monocarboxylate collector gave a high yield, on fluorite at the same pH both the monocarboxylate and the dicarboxylate collectors with one carbon between the carboxyl groups gave good results. On calcite at the natural pH the monocarboxylate collector was most efficient but the dicarboxylate collectors with a two- and a three-carbon spacer also gave a reasonable recovery. On fluorite at the natural pH the dicarboxylate collectors with a two- and a three-carbon spacer were most efficient. The potential and the flotation recovery of the mineral particles as afunction of added collector was assessed and the adsorption was also monitored by diffuse reflectance infra-red spectroscopy. Taken together, the results showed that small changes in the head group region of the collector can radically affect flotation recovery. This type of knowledge is important to understand flotation selectivity in a mixture of similar minerals.