In this study, the effect of different treatments including method 1- homogenized by-product (BP-FPI), method 2- washing of homogenized by-product by distilled water (WBP-FPI), method 3- washing of homogenized by-product by CaCl₂-citric acid (CaCi-BPFPI), method 4- CaCl₂-citric acid treated –washing by distilled water (CaCi-W- BPFPI), method 5- washing by distilled water- CaCl₂-citric acid treatment (W-CaCi-BPFPI) on composition of protein isolate from rainbow trout (Oncorhynchus mykiss) by-product were investigated. Washing by distilled water (method 2) and distilled water + combination of CaCl₂ + citric acid (method 5) had significant effect on heme iron and phospholipid (P < 0.05). Washing methods influenced protein yield and the lowest yield (9.4%) was obtained using method 5 (P < 0.05). Washing-derived proteins were lighter in color compared with that obtained from by-product. Method 1 resulted in higher redness (9.36) while that obtained with method 5 showed the lowest redness (1.19) (P < 0.05). Protein hydrolysate produced from method 5 protein isolate had high antioxidant activity in a dose-dependent manner.  Additionally, the presence of small peptides in hydrolysate contributed to its anti-diabetic activities. Results indicated that washing by distilled water (method 2) or combined with CaCl₂ + citric acid (method 5) is necessary for production of protein isolate from rainbow trout processing by-product.