Introduction: Pharmaceutical availability testing is one of the quality control methods used to assess the ability of a medicinal substance to diffuse from a substrate, through a membrane, into a suitable acceptor fluid. The most commonly used membranes for this type of testing are those made of polymers (cellulose, cellophane, or nylon membranes) or natural membranes (mouse/rat/pig skin, human cadaver skin). An alternative to the above membranes is the Strat-M® membrane, designed to mimic human and animal skin. Our study aimed to compare the pharmaceutical availability of insulin from a hydrogel matrix based on Sepineo™ P600 and Sepineo™ PHD100 (Seppic, Paris, France) using a Strat-M® membrane (Merck Millipore, Burlington, USA) and cellulose dialysis membrane Spectra/Por®2 (MWCO of 12-14 kDa; Spectrum Laboratories, Inc., California, United States). Methods: Insulin hydrogels (Insulatard Penfil) based on Sepineo™ P600 and Sepineo™ PHD100 were prepared. The insulin dose was set at 1mg/g. Pharmaceutical availability studies of insulin from hydrogel formulations were conducted in vitro in an ERWEKA DT600 paddle apparatus (Husenstamm, Germany), using Enhancer CellTM (Erweka, Husenstamm, Germany). The volume of PBS acceptor fluid was 50 mL. Testing was carried out at 32±1°C (human skin surface temperature). The stirrer speed was 100 rpm. The amount of insulin released was determined by spectrophotometry at λ = 271 nm. The Strat-M® membrane and cellulose dialysis membrane Spectra/Por® 2 were used in the study. Results: In a pharmaceutical availability study using the Strat-M® membrane, 53.36% and 47.4% of the API were released from the Sepineo™ P600 and Sepineo™ PHD100 hydrogel after 6.5 hours, respectively. In contrast, 69.6% and 64.3% of insulin were released through the cellulose dialysis membrane Spectra/Por®2 after 5 hours. The release of hormones from the hydrogel matrices followed the Peppas/Sahlin model. Conclusions: The amount of INS released by the Strat-M® membrane (high correlation with human skin) was lower than the cellulose dialysis membrane Spectra/Por®2.