Skeletal maturity assessment involves radiographic analysis and visual inspection of developing bone and their initial appearance or sequential ossification and related changes in size and shape along with the expression of various biomarkers in body fluids. To investigate the correlation of biomarkers such as salivary alkaline phosphatase (S-ALP) and salivary total protein (STP) with skeletal maturity assessment and growth prediction in growing children. A total of 8-15-year-old 150 healthy children were divided into five groups depending upon radiographic stage maturity of the middle phalanx of the left hand's third finger according to the Hagg and Taranger method. Radiographs were taken using intraoral periapical (IOPA) radiographic films. Salivary alkaline phosphatase (S-ALP) activity in the MP3 G group was significantly higher than MP3 F group and MP3 I group. Total protein levels in MP3 F were significantly lower than in MP3 G. The mean value of S-ALP (33541.45 IU/L) and that of STP (2.77 mg/mL) was observed to be highest in the MP3 G group (G3) group. Salivary total protein (STP) and S-ALP may be used as an additional diagnostic tool to assess skeletal maturation and optimize growth prediction during myofunctional orthodontic treatment. Skeletal maturity assessment plays a significant role in orthodontic diagnosis, treatment planning, and stability of orthodontic treatment. Radiographic parameters involve radiographic exposure; hence in this study noninvasive biomarkers such as S-ALP and STP have been evaluated for skeletal maturity assessment and growth prediction. Abhangi KK, Choudhari SR, Butala PB, et al. Salivary Total Protein and Alkaline Phosphatase Activity as Biomarkers for Skeletal Maturity and Growth Prediction in Healthy Children: An In Vivo Study. Int J Clin Pediatr Dent 2023;16(4):603-607.