Polyacrylamide gel electrophoresis, being the current method of choice for length determination of inorganic polyphosphate (polyP), requires a sequencing apparatus, relies on commercially not available polyP length standards and yields only a chain length distribution. State of the art polyP quantification involves enzymatic hydrolysis of polyP to orthophosphate with the Saccharomyces cerevisiae Exopolyphosphatase 1 (scPpx1p) and subsequent colorimetric orthophosphate detection. Because scPpx1p leaves one pyrophosphate per polyP, short chain polyPs are only partially detected. To overcome this analytical limitation, a method involving both the scPpx1p and the S. cerevisiae inorganic pyrophosphatase (scIpp1p) is proposed. Differential enzymatic hydrolysis of polyP with scPpx1p, and a combination of scIpp1p and scPpx1p allows not only for comprehensive quantification of polyP (excluding cyclic polyP) down to a chain length of two, but also absolute average chain length determination in the range of two to approximately 80. An optimized one-reagent method for rapid (2 min) orthophosphate quantification is part of the assay. Biological phosphorous containing molecules at equimolar phosphorous concentrations regarding polyP do not interfere. The method requires 1.5 μg polyP and calls only for a plate reader. This is the first enzymatic method for simultaneous average polyP chain length determination as well as comprehensive quantification.