In hair care cosmetic products' evaluation, one commonly used method is to evaluate the hair appearance as a gold standard in order to determine the effect of an active ingredient on the final state of the hair via visual appreciation. Although other techniques have been proposed for a direct analysis of the hair fibres, they give only surface or structural information, without any accurate molecular information. A different approach based on confocal Raman spectroscopy has been proposed for tracking in situ the molecular change in the keratin directly in the human hair fibres. It presents a high molecular specificity to detect chemical interactions between molecules and can provide molecular information at various depths at the cortex and cuticle levels.To evaluate the potential of confocal Raman spectroscopy in testing the efficiency of cosmetic ingredients on keratin structure, we undertook a pilot study on the effectiveness of a smoothing shampoo on natural human hair, by analysing α-helix and β-sheet spectral markers in the Amide I band and spectral markers specific to the cystin sulfur content.We confirmed that an active proved to be effective on a gold standard decreases α-helix keratin conformation and promotes β-sheet keratin conformation in the hair fibres. We also showed that treatment with the effective active decreases the intensity of covalent disulfide (S-S at 510 cm ) cross-linking bands of cysteine. These data confirm that the effective active also acts on the tertiary structure of keratin.From these experiments, we concluded that the effective active has a smoothing effect on the human hair fibres by acting on α-helix and β-sheet keratin conformation and on the tertiary structure of keratin. Based on these results, confocal Raman spectroscopy can be considered a powerful technique for investigating the influence of hair cosmetic ingredients on keratin structure in human hair fibres. Moreover, this analytical technique has the advantage of being non-destructive and label free; in addition, it does not require sample extraction or purification and it can be applied routinely in cosmetic laboratories.