Bivariant multiple development thin-layer chromatography technique (BMD–TLC) along with high-performance liquid chromatography–diode array detection–electrospray ionization–mass spectrometry (HPLC–DAD–ESI–MS) analysis was used in determination of lutein or lutein mixed with zeaxanthin in eight dietary supplements. The developed two-step TLC separation procedure combined purification, compaction of samples and separation of the analyzed compounds what significantly shortened and simplified samples preparation. Qualitative analysis was based on co-chromatography with reference substances and HPLC–DAD–ESI–MS analysis. It was revealed that three of eight dietary supplements did not contain lutein. In turn, quantitative analysis with the use of developed TLC conditions along with densitometry showed that the amount of lutein or its mixture with zeaxanthin in the others differed from that claimed by producers.