The purpose of determining serum cortisol level is to reflect the activity of stress axis, ethological alterations, acute and chronic pain, life quality, or psychogenic stress. Although it is stated that stress can produce a measurable influence on the cortisol level, a certified value of this pain biomarker in dogs was not generally accepted yet. This study aimed to investigate if serum cortisol measured follows allopathic treatments only, or it is associated with physiotherapy, point out pain level in dogs with orthopedic disease, which could reveal the healing progress. The diagnostic identified: hip dysplasia, cranial cruciate ligament rupture, or intervertebral disc disease. Ortolani and Barden tests, together with clinical examination, drawer sign, and tibia compression test, were done in dogs exhibiting postures, and motion alteration, and X-Ray confirmed. A total of 30 dogs were grouped in healthy (n = 10) and pain groups (n = 20), the blood sampling is done at the beginning of the investigation, and after ten days of the study. Dogs were handled in two ways: G1—treated with Nonsteroidal Anti-Inflammatory Drugs (NSAIDs) only and respectively, G2—by therapy and physiotherapy. The analysis was performed on a Roche Cobas Analyzer (Roche, USA), serum cortisol being determined by Electrochemiluminescence immunoassay (ECLIA), and statistics using ANOVA, following Tukey’s Multiple Comparison Test. The results revealed that, out of ten specimens in the Control group, nine were within the normal limits: 5–65 ng × mL−1 (24.76 ± 19.48678), and one sample under the set limit. In G1, it was observed that the plasmatic P1 values were below the levels of P2, in six situations. In G2, although the status of all subjects improved radically with the removal or evident reduction of pain, confirmed clinically and imagistically, the P2 values in five dogs were higher than the initial P1 values, and in contradiction with the observed clinical reality. Comparing results, the mean difference in G1 was 0.41, and in G2 = 2.54, with an SD for G1 = 13.38, and G2 = 16.66, registering moderate development. Standard deviation illustrated that the values of treated groups were highly spread throughout the interval, and the serum cortisol assay did not generate significant statistical differences between groups in our case. This inferred the doubt whether the used detection method or values registered correctly indicates the pain levels in dog species.