John Glassford was recognised by contemporaries as the greatest of Glasgow's tobacco lords. Tobias Smollett, the novelist, described him as "one of the greatest merchants in Europe", who was "accustomed to lay out his money freely on bold adventures in trade". His father, James, was a Paisley merchant and John cut his commercial teeth in the textile trades. By the 1740s he had interests in a number of manufactories including the Stocking Manufactory Company and the Glasgow Inkle Factory.
He did not begin trading in tobacco until 1750 and quickly built up a fleet of twenty-five vessels and a string of stores across New England with nine in Maryland alone. In the year following the outbreak of the American War of Independence he personally traded in 1,823 hogsheads of tobacco. He retained his interest in textiles, becoming a partner in the Pollokshaws Printfield Company in 1761 and like other Tobacco Lords became involved in banking, helping to establish the successful Thistle Bank in 1761.
He invested heavily in land, buying property in Ayrshire, Lanarkshire and Stirlingshire. His country seat was at Netherwood to the north of Glasgow, which he renamed Dougalston, and much improved to the extent that the fields looked like gardens.
He married as his second wife, Margaret, daughter of the 3rd Earl of Cromartie. He died just before the coming of peace with America and it was left to his executors to sort out his tangled affairs and lay claims for compensation for losses in America with the British Government.