AbstractBackgroundWe aimed to develop a single step method for the production of human platelet lysate (hPL). The method must result in high hPL yields, be closed system and avoid heparin use.Study Design and MethodsThe method aimed at using glass beads and calcium. An optimal concentration of calcium and glass beads was determined by serial dilution. This was translated to a novel method and compared to known methods: freeze‐thawing and high calcium. Quality outcome measures were transmittance, fibrinogen and growth factor content, and cell doubling time.ResultsAn optimal concentration of 5 mM Ca2+ and 0.2 g/ml glass beads resulted in hPL with yields of 92% ± 1% (n = 50) independent of source material (apheresis or buffy coat‐derived). The transmittance was highest (56% ± 9%) compared to known methods (<39%). The fibrinogen concentration (7.0 ± 1.1 μg/ml) was well below the threshold, avoiding the need for heparin. Growth factor content was similar across hPL production methods. The cell doubling time of adipose derived stem cells was 25 ± 1 h and not different across methods. Batch consistency was determined across six batches of hPL (each n = 25 constituting concentrates) and was <11% for all parameters including cell doubling time. Calcium precipitation formed after 4 days of culturing stem cells in media with hPL prepared by the high (15 mM) Ca2+ method, but not with hPL prepared by glass bead method.DiscussionThe novel method transforms platelet concentrates to hPL with little hands‐on time. The method results in high yield, is closed system, without heparin and non‐inferior to published methods.