Upon subcutaneous (SQ) injection of therapeutic drugs, a local inflammatory reaction, commonly referred to as an injection site reaction (ISR), may develop at the injection site. ISRs are characterized by one or more of the following, erythema, edema, pruritus, pain, and induration. Therefore, ISRs are highly undesirable as they result in a suboptimal patient experience and represent a significant barrier for patient compliance, particularly for frequently administered therapeutics. Despite a well-recognized and long-standing unmet need, the development of validated model systems that can enable the identification of safer therapeutic candidates that have favorable ISR profiles has proven to be elusive. Here we use standardized immunocompetent human skin explants from live donors containing epidermis, dermis, and hypodermis to enable subcutaneous injection (up to 100 µL), to assess the extent to which therapeutic molecules trigger an ISR-like inflammatory response at the site of injection. To enable broad utility agnostic to therapeutic molecule classes, mechanism of action, and stage of development, we leveraged two complementary readouts: multiplex cytokine profiling to broadly survey pro-inflammatory responses and mast cell degranulation by immunofluorescence of whole skin sections. Compared to skin explants injected with vehicle control (phosphate buffered saline), the injection of the Mas-related G-protein coupled receptor member X2 (MRGPRX2) agonist compound 48/80, or Kineret (i.e., a commercial biologic that is a known competitive inhibitor of the IL-1R1 and triggers intense ISRs in up to 70% of patients) induced statistically significant and reproducible exteriorization of mast cell cytoplasmic granules. Moreover, we observe a dose-response of mast cell degranulation upon injection with different concentrations of compound 48/80 in the tissue. Further, SQ injections of molecules known to cause ISRs in the clinic produced a pro-inflammatory cytokine response assayed by multiplex profiling that is consistent with the clinical observation. Our results strongly suggest that these standardized human skin explants from multiple donors, maintained in an immunocompetent state, can be used for the identification and selection of safer and better tolerated therapeutics by predicting the extent to which candidate drugs can trigger ISRs when injected in patients.