Heterotrophic flagellates contribute significantly to the matter flux in aquatic and terrestrial ecosystems. Still today their quantification and taxonomic classification bear several problems in field studies, though these methodological problems seem to be increasingly ignored in current ecological studies. Here we describe and test different methods, the live-counting technique, different fixation techniques, cultivation methods like the liquid aliquot method (LAM), and a molecular single cell survey called aliquot PCR (aPCR). All these methods have been tested either using aquatic field samples or cultures of freshwater and marine taxa. Each of the described methods has its advantages and disadvantages, which have to be considered in every single case. With the live-counting technique a detection of living cells up to morphospecies level is possible. Fixation of cells and staining methods are advantageous due to the possible long-term storage and observation of samples. Cultivation methods (LAM) offer the possibility of subsequent molecular analyses, and aPCR tools might complete the deficiency of LAM in terms of the missing detection of non-cultivable flagellates. In summary, we propose a combination of several investigation techniques reducing the gap between the different methodological problems.