High-performance liquid chromatography is considered as the selected analytical tool for a huge number of applications, including the carotenoid analysis. However, due to the great complexity of some natural samples containing this kind of compounds, conventional LC could not have enough separation power. In this work, serial connection of several columns is proposed as an alternative to LC and also to comprehensive two-dimensional LC for the analysis of carotenoids from complex real matrices. Different parameters regarding these separation procedures are studied and discussed, such as the stationary phase used or the application of high separation temperatures. The applicability of connecting two C30 columns to significantly increase the separation power, resolution and peak capacity for the analysis of carotenoids has been demonstrated for the first time. In fact, a peak capacity of 79 was obtained when using two C30 serial coupled columns, compared to 61 achieved using a single column. Similar improvements were also observed for the other serial couplings studied. The present methodology could be applied to the analysis of carotenoids in a great variety of samples. To the best of our knowledge, this is the first development carried out to study natural products such as carotenoids using columns coupled in series.