Background: Baricitinib (BCTB) is a novel Janus Kinase (JAK) 1 and 2 inhibitor used in the therapy of rheumatoid arthritis, approved by the “Food and Drug Administration” in 2018. It has significant dose-dependent effectiveness and severe side effects. Thus, it is crucial to figure out its concentration in developed dosage forms. The literature search revealed that there has only been one UV spectroscopy technique documented up to this point. Methanol was chosen as the detection medium in this approach, which is not comparable with plasma or serum. As a result, the preliminary research suggested developing a UV spectroscopic approach that can estimate BCTB concentration and compare it to its concentration in the plasma or serum. Thus, in the proposed method, 7.4 pH phosphate buffer was selected as a mobile phase. Aim: Using the Analytical Quality by Design (AQbD) methodology, a simple, robust spectrophotometric method for the detection of BCTB in API form and Niosomes drug delivery system is designed and assessed. Methods: In the AQbD approach, a face-centered CCD design of Design Expert 13 software was used to evaluate two critical method variables: scanning speed and sampling interval. The design space suitability was confirmed by standard error and overlay plots. The 2-D contour and 3-D response surface plots were used to forecast the relationship between the response variable and predictor variables. Results: Baricitinib displays an absorption maximum at 249.40 nm in saline phosphate buffer pH 7.4. The distinguished linearity of the method was obtained over a concentration of 5–30 µg/ml with a correlation coefficient (R2 ) value of 0.998. BCTB % assay was found to be near 99 %. Intraday and Interday precision were found to have % RSDs of 0.067–0.488 and 0.146–0.942, respectively. Conclusion: The established spectrophotometric technique was observed to be precise as per ICH revised guidelines ICH Q2 (R1) and Q14 for analytical method validation. Our findings are instructional for the future design and development of safe and reliable therapeutic dosage forms of BCTB for rheumatoid arthritis.