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Background and Objectives The degree of protective cooling required during laser therapy to achieve an optimal result is unknown. The expression of heat shock proteins, Hsp47 and Hsp70, were examined in the epidermis and dermis as biomarkers to quantify the degree and depth of tissue affected by non-ablative laser treatment using variable protective cooling parameters. Study Design/Materials and Methods Twenty-one male Sprague–Dawley rats were treated with a 1,319 nm Nd:YAG laser using a sapphire cooling plate attached to the hand piece. A 4 cm×4 cm area on each side of the rat was treated with the same energy and pulse settings, with variable contact cooling. Protective cooling parameters, for each degree increment, ranging from 0 to 25°C were studied. Immunohistochemistry (IHC), Western blot and PCR were performed to evaluate the effects of superficial cooling on Hsp47, and Hsp70 expressions. Results Our data showed the extent of topical cooling needed to produce a thermal effect at different depths in the dermis, quantified by the expression of Hsp47 and Hsp70. Significant Hsp expression was observed with cooling of 13°C and warmer; no identifiable cellular reaction was observed when cooling below 5°C. There was no evidence of epidermal injury when treating the skin with any protective cooling ranging from 0 to 25°C. Conclusion Our data would suggest contact cooling temperatures 5°C and below completely protects through the entire dermis. There was no evidence of epidermal injury with protective cooling at any temperature between 0 and 25°C. Warmer temperatures are safe and adequately protect the epidermis in this model. Lasers Surg. Med. 39:504–512, 2007. © 2007 Wiley-Liss, Inc.