A British Foreign and Commonwealth Office spokesman said: "The Royal Navy challenges all unlawful maritime incursions into British Gibraltar Territorial Waters - and did so again on this occasion".
It comes after Lord Howard suggested that Britain should "go to war" over Gibraltar, in a similar vein to Margaret Thatcher's reaction to the Falklands conflict with Argentina.
The former Tory leader was speaking following the unveiling of EU Brexit negotiation guidelines which suggest Spain could have a veto over any future trade deal with the United Kingdom unless it gets an input into the territory's future following the divorce.
"Gibraltar's sovereignty has not changed, it will not change and can not change without the consent of the people of Gibraltar and the United Kingdom".
However she's believed to have phoned Gibraltarian chief minister Fabian Picardo to assure him of her support.
"It's not so much that Spain will take Gibraltar over, or have a much more serious threat on British sovereignty in Gibraltar, but rather that the economic basis of Gibraltar is threatened", said analyst Andrew Canessa.
Mr Picardo said: "Let us be very clear and let the message be clear in Madrid, in Brussels and in every other capital of the European Union".
Lord Howard told Sophy Ridge's Sunday morning politics show on Sky that 35 years ago: "Another woman Prime Minister sent a taskforce halfway across the world to protect another small group of British people against another Spanish-speaking country".
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He acknowledged that Brexit could cause difficulties in the relationship with Spain, and criticised the European Union for the "rather unsafe and slightly risky" decision to mention Gibraltar in the draft negotiating text "because it is likely to raise the temperature in what is going to be a hard negotiation".
The British government triggered Article 50 of the Lisbon Treaty and started official Brexit negotiations on March 29.
Spanish foreign minister Alfonso Dastis criticised Lord Howard's comments, saying: "Comparing Gibraltar with the Falklands is taking things out of context".
Speaking on British radio, the former Labour Party foreign secretary Jack Straw said he did not see Gibraltar as being any kind of deal breaker in the negotiations.
Gibraltarians voted overwhelmingly in last June's referendum to stay as part of the European Union but Britain's decision as a whole to leave potentially takes the overseas territory with it. "Removal of the reference to Gibraltar would be a sign of good faith and good will", he said. Gibraltarians rejected co-sovereignty by 99% to 1%.
"I think at the end of the day, it is that which will win through".
Spain, which has for centuries demanded a return of sovereignty over the enclave on its southern tip, doesn't recognize Gibraltar's territorial waters.
Asked again about Howard's remarks, the spokesperson added: "What Lord Howard was trying to establish was the resolve that we will have to protect the rights of Gibraltar and its sovereignty".
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