Monitoring cytokine profiles plays a crucial role in predictive and early disease diagnosis, as well as in research in many biological fields; thus, multiple approaches to detect cytokines have been investigated. Despite the promise of these techniques, many still require specialized instrumentation or have not been demonstrated in real-time applications, impeding their clinical adoption as point-of-care systems. Impedance-based protein detection sensors can achieve quantitative specificity and rapid or real-time operations, potentially enabling point-of-care diagnostics. Furthermore, microfabrication of these sensors can lead to form factors suitable for in vivo applications. Herein, we present microfabricated, needle-shaped and microwell-based impedance sensors for label-free, rapid sample-to-answer, real-time and in vivo cytokine detection. A microwell array was configured on the microneedle tip to enable label-free detection while maintaining high sensor sensitivity, despite the high salt concentration of complex biological fluids. The microneedle form factor allows for utilization in transcutaneous or transvascular sensing applications. In vitro characterizations confirmed sensor specificity and sensitivity to multiple cytokines of interest.These sensors were tested with an animal model system, consisting of normal mice and transgenic mice that endogenously expressed human interleukin 8 (hIL8), to evaluate their potential for real-time cytokine quantification in vivo. Needle-based sensors were inserted directly into the blood and/or muscle of transgenic mice. Sensor responses were subsequently compared to the hIL8 concentration levels in serum extracted from the same mice as quantified by ELISA. Excellent agreement between these two methods was observed over multiple transgenic mice expressing hIL8 concentrations from 62 pg/mL to 539 ng/mL. Further improvements in sensor form factors and functionality were also investigated. To reduce the large mechanical mismatch between inorganic microneedles and surrounding soft tissue for potential chronic applications, materials with lower Young’s modulus were employed as the sensor substrate. Moreover, the relative dimensions of the microwell array and microneedle offer the potential for multiple bioassay sensors on a single microneedle. By forming such multiple sensors on a single microneedle and functionalizing the surface of each sensor with distinct antibodies, a multiplex-type sensing platform for multiple cytokines was realized, allowing for the real-time assessment of more complex diseases or conditions in vivo.