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Preventing separation-related behaviors (SRBs) is important for canine welfare, potentially reducing relinquishment or euthanasia. This double-blind study assessed efficacy of advice provided to prospective puppy owners. Thirty-four owners were randomly assigned to treatments: (1) Control, receiving responsible ownership advice; (2) Calm, receiving control advice plus advice on calm interactions during departures and reunions; (3) Habituation, receiving control plus advice on gradually increasing time left alone; or (4) Combination, receiving all advice. Owners filmed puppies alone at four timepoints over 6 months and during a separation test. A treatment-blind observer scored puppy behavior, later categorized as ‘active anxious’ (e.g. whining, barking), ‘passive anxious’ (e.g. panting, lip-licking), ‘inactivity’ (e.g. lying) and ‘active positive’ (e.g. playing, eating). Recruitment challenges caused sub-optimal sample size, rendering findings exploratory. Calm group puppies exhibited more inactivity than Control and Combination across timepoints and in separation tests; Habituation group puppies showed more inactivity than Controls during separation tests. Other behavior categories showed no significant associations with treatment. However, across treatments, puppies whose owners did more habituation activities showed less passive anxious behavior. Passive anxious scores were greater in puppies left alone than with other dogs. When radio or TV was on, versus off, puppies showed more passive anxious and less active positive behavior. While the advice did not significantly reduce SRBs, possibly due to sub-optimal sampling, the Calm advice, and to some extent Habituation, were associated with greater inactivity when dogs were alone. Further research could aim to verify findings and maximize owner compliance with SRB prevention advice. • Owners received differing advice to prevent puppy separation related behavior (SRB) • Videos of puppies left alone at different ages were scored by a treatment-blind observer • Advice for owners to behave calmly was associated with puppy inactivity when alone • Advice to habituate puppies to be alone also increased puppy inactivity when alone • However, advice did not significantly reduce active SRBs and reasons are discussed