Transporter proteins are essential across the tree of life as they provide a cell with a means of exchanging vital metabolites with the external milieu. Characterizing the function of transporters is challenging and traditionally uses methods involving radiolabelled substrates, which requires prolonged exposure times and specialist equipment. Here, we provide an alternative method to the classical uptake assay using Raman spectroscopy to detect the uptake of alkyne-labelled substrates and determine transporter function. As a proof of principle, we demonstrate the method using a candidate nucleotide transporter (ThNTT4) expressed in