The analysis of beta-carboline alkaloids presents a renewed interest due to their biological relevance and their increasing popularity as recreational drugs. In the present work, a novel chromatographic reverse-phase high- performance liquid chromatography (RP-HPLC) method with fluorimetric detection has been applied to the determination of beta-carbolines spiked in human serum samples. The chromatographic procedure involves the use of less retentive, unusual C1 columns combined with hydro-alcoholic mobile phases and the use of beta- cyclodextrin or (2-hydroxypropyl)-beta-cyclodextrin as mobile-phase additives. The effective combination of C1 columns and the modified mobile phases with cyclodextrins leads to a considerable reduction in the organic proportion in the mobile phase (up to 50%) with good resolution and efficiency. Besides, the presence of cyclodextrins allows the use of ethanol, a green solvent, as the organic component in the mobile phase. Traditional RP-HPLC thus becomes an attractive eco-separation technique using conventional stationary phases under simple and user-friendly experi- mental conditions. Solid-phase extraction was employed as sample clean-up protocol with attractive features, i.e., a low consumption of organic solvents, time and step economy and diminished need for sample handling. The analytical procedure was completely validated showing satisfactory figures of merit. Limits of detection of 10−9–10−10 M can be achieved. The recoveries obtained for the total method- ology (sample pre-treatment and chromatographic determi- nation in the case of the mobile phases containing cyclodextrins) were very satisfactory (95–107%) as well as the intraday (2–3%) and interday precision values (3– 7%). The use of 3-hydroxymethyl-beta-carboline as an internal standard allows the comparison of the goodness of response of the analytical methodology in the presence or absence of cyclodextrins. Keywords Cyclodextrins . Eco-friendly mobile phases . β- carboline derivatives . HPLC