Urinalysis, a common test in infants, could represent a suitable non-invasive clinical tool in puppies. In dog neonates, urine is easily collected by stimulating the somato-vesccal reflex. Information on urine characteristics during the neonatal period is missing. Beside instrumental laboratory analyses, the dipstick was proven useful for rapid urinalysis to evaluate specific gravity (SG), pH, leukocytes, nitrites, glucose, proteins, ketones, urobilinogen, bilirubin, and blood. The present study aimed to describe urinalysis features by the dipstick test and refractometer along the neonatal period. Urine samples (n = 624) were collected by manual stimulation from 48 healthy Great Danes, daily from birth to seven days, then twice a week until 28 days, to assess age-related changes (ANOVA, p < 0.05) and the possible effects of gender and litter (T-test, p < 0.05). The SG and pH significantly changed during the neonatal period. Other parameters did not vary significantly in relation to age. No significant differences were observed either among litters or between genders. The present study confirmed that canine kidneys are able to concentrate urine from the second week of age when the urinary SG started to be similar to adults, while pH still increased towards the typical values of adults at 28 days. Significant glucosuria and proteinuria were never detected. Dipstick urinalysis represents a useful first-line complementary tool in newborns clinical examination, providing information about systemic homeostasis.