Three simple, sensitive and robust kinetic spectrophotometric methods for the determination of a novel lipophilic thiol compound, N-acetyl-L-cysteine ethyl ester (NACET), have been developed and validated. The methods are based on the reduction of Cu(II)-ligand complex to Cu(I)-ligand complex with the analyte. Studied ligands were neocuproine, bicinchoninic acid (BCA) and bathocuproine disulfonic acid (BCS). The development of chromogenic complexes was followed using kinetic setup with spectrophotometric detection at 458, 562 and 483 nm for the reactions of NACET with neocuproine, BCA and BCS, respectively. The calculated reaction orders with respect to NACET concentration were found to be 1.07, 1.01, 1.07, respectively, thus confirming a first order of reaction. The initial rate and fixed time methods were utilized for constructing calibration curves. Assays limits of detection were 1.4 × 10–7, 3.2 × 10−7 and 6.0 × 10−8 mol L–1, respectively. The analytical performance of the methods, in terms of accuracy and precision, was established.