: The development of a five-in-one vaccine microneedle patch (five-in-one MN patch) aims to address challenges in administering vaccines against Diphtheria (DT), Tetanus (TT), Pertussis (wP), Hepatitis B (HBsAg), and type b (Hib). Combining multiple vaccines into a single patch offers a novel solution to improve vaccine accessibility, stability, and delivery efficiency, particularly in resource-limited settings. : The five-in-one MN patch consists of four distinct microneedle arrays: DT and TT vaccines are coated together on one array, while wP, HepB, and Hib vaccines are coated separately on individual arrays. The patch was tested for long-term stability (12 months at 25 °C) and evaluated for immunogenicity in mice and minipigs. Antibody titers were measured using ELISA to compare immune responses between microneedle-based delivery and traditional intramuscular (IM) injection. : The five-in-one MN patch demonstrated stable antigenicity for up to 12 months at room temperature. In animal studies, the patch induced antibody titers comparable to traditional IM injections for all vaccines. Notably, immunogenic responses to Pertussis and type b vaccines via microneedles were reported for the first time. The patch facilitated the simultaneous yet independent delivery of vaccines, preserving their immunogenicity without interference. : The five-in-one MN patch represents a significant advancement in vaccine delivery by enabling stable, minimally invasive, and efficient immunization. Its innovative design addresses the critical limitations of combination vaccines and has the potential to enhance vaccine accessibility in low- and middle-income countries. Future studies will focus on optimizing patch application techniques and evaluating broader clinical applicability.