Astaxanthin is a potent natural antioxidant with beneficial bioactivities demonstrated primarily for its free (non-esterified) form. However, its natural producer, the microalgae Haematococcus pluvialis synthesizes astaxanthin mostly in ester forms which have been little valorized so far. Hence, to contribute to the commercial use of astaxanthin esters, a scalable and efficient isolation technology is required. In this study, five astaxanthin monoesters were isolated from H. pluvialis using high performance countercurrent chromatography (HPCCC), where the lower phase of a biphasic solvent system (n-heptane:acetonitrile, ratio 5:5, v/v) was used as a mobile phase. Around 200 mg of biomass extract was subjected to the HPCCC leading to a separation of the target astaxanthin esters. To further increase the process productivity, a multi-injection HPCCC method was developed by combining two elution modes (reverse phase and co-current). In co-current elution mode, both the mobile and stationary phases were pumped simultaneously at flow rates of 3 and 1 mL/min respectively, so that the stationary phase that gets lost during each separation cycle is replenished. In total, five injections of samples (200 mg of extract, each) were achieved. Final purification with high performance liquid chromatography (HPLC) afforded five astaxanthin derivatives esterified with α-linolenic acid (1, 4 mg), linoleic acid (2, 8 mg), palmitic acid (3, 8 mg), oleic acid (4, 12 mg) and stearic acid (5, 1 mg) with purities of 98%, as determined by HPLC analysis. Only compound 4 exhibited a cytotoxic effect against human gastric cancer cells. The present study shows a useful approach for obtaining individual astaxanthin esters from H. pluvialis.