Biotherapeutics is the fastest growing class of drugs administered by subcutaneous injection. In vitro release testing mimicking physiological conditions at the injection site may guide formulation development and improve biopredictive capabilities. Here, an in vitro release cartridge (IVR cartridge) comprising a porous agarose matrix emulating subcutaneous tissue was explored. The objective was to assess effects of medium composition and incorporation of human serum albumin into the matrix. Drug disappearance was assessed for solution, suspension and in situ precipitating insulin products (Actrapid, Levemir, Tresiba, Mixtard 30, Insulatard, Lantus) using the flow-based cartridge. UV–Vis imaging and light microscopy visualized dissolution, precipitation and albumin binding phenomena at the injection site. Divalent cations present in the release medium resulted in slower insulin disappearance for suspension-based and in situ precipitating insulins. Albumin-binding acylated insulin analogs exhibited rapid disappearance from the cartridge; however, sustained retention was achieved by coupling albumin to the matrix. An in vitro-in vivo relation was established for the non-albumin-binding insulins. The IVR cartridge is flexible with potential in formulation development as shown by the ability to accommodate solutions, suspensions, and in situ forming formulations while tailoring of the system to probe in vivo relevant medium effects and tissue constituent interactions.