•Hematocrit issues still prevent full acceptance of dried blood spot analysis.•Many attempts to overcome this hematocrit bias have been proposed.•Whole spot analysis with volumetric application of blood should nullify this bias.•Devices generating dried plasma spots may avoid all hematocrit-related issues.•Strategies to measure the hematocrit of a dried blood spot have been proposed. Hematocrit-related issues remain a major barrier for (regulatory) acceptance of the classical dried blood spot (DBS) analysis in the bioanalytical and clinical field. Lately, many attempts to cope with these issues have been made. Throughout this review, an overview is provided on new strategies that try to cope with this hematocrit effect (going from avoiding to minimizing), on methods estimating a DBS volume, and on methods estimating or measuring the hematocrit of a DBS. Although many successful strategies have been put forward, a combination of different technologies still provides the most complete solution. Therefore, further efforts and the availability of a straightforward guideline for analytical and clinical method validation should help to overcome the hurdles still associated with DBS sampling.