A recent development in algal pigment analysis by high-performance liquid chromatography (HPLC) is the application of automation. An optimization of a complete sampling and analysis protocol applied specifically in automation has not yet been performed. In this paper we show that automation can only be successful if the various methodological aspects of the sampling and analysis protocol are considered in coherence. We introduce an optimized protocol that involves freeze-drying of the sample, subsequent extraction in 90% acetone and the application of water-packing during analysis. The method was evaluated on both natural plankton populations and a broad spectrum of microalgal cultures: Thalassiosira weisflogii (Bacillariophyceae), Emiliania huxleyi (Prymnesiophyceae), Phaeocystis globosa and Phaeocystis antarctica (Prymnesiophyceae) and Pyramimonas sp. (Prasinophyceae). Whereas pigment extracts were unstable in methanol, with recorded chlorophyll a losses from 10% to 60% per day, pigment degradation rates in acetone were generally less than 1% over 18 h storage in the autosampler (4 ยฐC). In addition, it was found that the extraction efficiency of acetone significantly increased upon freeze-drying prior to extraction. Increases as high as 50-60% were measured in P. antarctica. The application of water-packing of the sample during injection resulted in improved peak shape and peak separation, without diluting the pigment concentrations. Automation is especially beneficial for application in the field, when mixed algal assemblages and low biomass put a high demand on the sensitivity as well as reproducibility of the method.