•We set up a culture-independent assessment of soil microbial biomass.•PicoGreen quantification of crude dsDNA extracts provides reliable data.•The method is simple, high-throughput, and does not require expensive equipment. We set up a simple, culture independent, low-cost and high-throughput method for DNA-based quantitative assessment of soil microbial biomass using eight soils covering a wide range of physico-chemical properties. DNA was extracted with a 0.12M, pH 8 Na2HPO4 buffer using bead beating; double stranded DNA (dsDNA) was quantified in a crude (not purified) extract using PicoGreen reagent. In contrast to yields obtained by using a commercial standard method (FastDNA Kit for soil, MP-Biomedicals), our yields of dsDNA were generally higher, most probably because any purification method for obtaining highly pure DNA for downstream analyses leads to DNA loss. These results suggest the new method provides more reliable quantitative data; thus it is a good environmental indicator, as an underestimation of the soil microbial biomass due to DNA loss during purification can be excluded. The ratio between microbial C (Cmic) obtained by the traditional, widely used fumigation-extraction method and dsDNA ranged from 12.0 to 63.5μg Cmic per μg dsDNA. Crude DNA obtained by the new method as well as purified DNA obtained by using the commercial kit were compared in terms of quantity (fluorometry; spectrophotometry) and quality (purity indices: A260/A280, A260/A230; PCR compatibility; gel electrophoresis: molecular weight and molecular integrity). Our results suggest that the new method provides a high-throughput estimator of microbial biomass (expressed as μgdsDNAg−1 soil) in soils having widely different properties without the need for high-cost commercial extraction kits and/or cumbersome individual methods. Due to its simplicity, speed and low-cost, our method is capable for routine quantitative assessments of soil microbial biomass, assessable also for soil scientists with laboratories that are otherwise not equipped for molecular analyses.