Simple SummaryThe multiple implications of serotonin in behavior manifestations have shaped the goal of the present study, which was to evaluate the variation of serum serotonin levels in different experimental groups of dogs to establish whether serum serotonin levels could serve as indicators of aggressive behavior, especially when adoption is considered. The experimental groups were divided into three variants: Variant 1—two groups of medium (n = 6) and small (n = 4) breed shelter dogs; Variant 2—dogs with owners (n = 15) and dogs without owners but in foster care (n = 10), after administration of pre-spaying/neutering anesthesia; and Variant 3—dogs in different behavioral states (n = 8), classified as follows: M1—happy, M2—aggressive, M3—calmed status, post-exposure to a stressful situation, compared to the reference time referred to as M0. Significant results were found between M1 and M2 (p ≤ 0.05, decrease of serotonin by 89.61 ng/mL), as well as between M2 and M3 (p ≤ 0.008, increase by 112.78 ng/mL). Following anesthesia, the average mean serotonin values were significantly lower (p ≤ 0.003), by 63.85 ng/mL, in stray dogs compared to dogs with owners, leading to a presumptive conclusion that serotonin levels could serve as indicators for potentially aggressive behaviors.Serotonin is considered to be the neurotransmitter that controls several types of behavior: aggressiveness, impulsivity, food selection, stimulation, sexual behavior, reaction to pain, and emotional manifestations. The aim of this study was to determine the serotonin values in 43 dogs, divided into three different experimental variants: (1) between two groups of medium (n = 6) and small (n = 4) breed shelter dogs; (2) in dogs with (n = 15) and without (n = 10) owners after administration of pre-spaying/neutering anesthesia; (3) in different behavioral states (n = 8) classified as follows: M1—happy, M2—aggressive, M3—calmed status, post-exposure to a stressful situation, compared to the reference time referred to as M0. There were no significant differences (p ≥ 0.05) regarding the serotonin values between the two groups of medium and small breed shelter dogs. Following anesthesia, the average mean serotonin values were significantly lower (p ≤ 0.003), by 63.85 ng/mL, in stray dogs compared to dogs with owners. No significant differences (p ≥ 0.05) were found when comparing the reference time M0 to M1, M2, and M3. The differences decreased significantly (p ≤ 0.05), by 89.61 ng/mL, between M1 and M2 and increased significantly (p ≤ 0.008), by 112.78 ng/mL, between M2 and M3.