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Jyoti Subhash started her career through theatre and then moving to television and films. She was recognised in her early works of television. Aired on Doordarshan, she featured in the telefilms Rukmavati Ki Haveli (1991) and Zazeere (1992). Directed by Govind Nihalani, the 1991 show Rukmavati Ki Haveli was based on the Spanish play The House of Bernarda Alba which was written by Federico García Lorca. A story of a new-widow, Rukmavati, raising her five unwed daughters in her haveli in Rajasthan, was shot on a 16 mm film and later blown up to 35 mm. Recently in 2009, the film was shown in a special session by National Centre for the Performing Arts (NCPA), Mumbai. In 1999, she translated the Marathi play Raste, originally written by Govind Purushottam Deshpande into Hindi as Raaste. The Hindi play was directed by Arvind Gaur and Satyadev Dubey. She played various supporting roles of elder women in the family in films like Dahavi Fha, Devrai, Aamhi Asu Ladke, Shubhra Kahi and more.

In 2004, she was seen in an Urdu play Jis Lahore Naee Dekhya about a story based in the partition era of India. Subhash played the role of an aged Hindu woman left behind in Lahore while her family migrates to India. Her haveli is then occupied by a Muslim family who at first are hostile with her but later on accept her into their family.

In 2006, she acted in a Marathi Movie “NITAL” directed by Sumitra Bhave and Sunil Sukhthankar. Neena Kulkarni was a co-actress. Lead character was played by Devika Daftardar. Film was produced by Dr. Maya Tulpule, founder and president of 'Shweta Association', a Vitiligo Self Help Support Group. Film showcased the story of a girl having vitiligo and social stigma around it.

Directed by Umesh Vinayak Kulkarni and with an ensemble cast, Valu (2008) was a comedy film where she played Sakhubai. The film also starred Girish Kulkarni in a major role, along with other actors like Atul Kulkarni, Mohan Agashe, Dilip Prabhavalkar, Nirmiti Sawant and more. The script was written by Umesh and Girish Kulkarni. They together made their next film, Vihir, in 2010 where again Subhash was cast. She played the role of a grandmother to two school going boys who come to their village in their holidays. Next year in 2011 the Kulkarni duo came up with the satirical film Deool where again Subhash was included. Girish Kulkarni considers her to be their "lucky mascot". She has also played Kulkarni's mother in Gabhricha Paus where the plight of Vidarbhian farmers was depicted. In 2009, she played the role of a mother worried about her daughter's marriage in the film Gandha. Directed by Sachin Kundalkar, the film had three different stories and Subhash played her real-life daughter, Amruta Subhash's mother. In 2012, the film was made in Hindi by Kundalkar as Aiyyaa where she played the lead actresses's grandmother; a role which was not present in the original Marathi version. The lead role was played by Bollywood actress Rani Mukerji while her mother was played by Nirmiti Sawant. Subhash's role was of comedy genre as a wheel-chair ridden grandmother and was considered delightful.

In Masala (2012), she plays the supporting role of a wife of a businessman (played by Mohan Agashe) who helps the lead character played by Girish Kulkarni set up his new business. Recently in 2013, she was part of the Marathi play Uney Purey Shahar Ek (or Boiled Beans on a Toast), originally written by Girish Karnad in Kannada as Benda Kaalu on Toast. Being story of a city, rather than of people, the play had cast of Radhika Apte, Vibhavari Deshpande, Anita Date, Ashwini Giri and more.^[11][12][13]

• Note: Unless otherwise noted, below works are in Marathi language.

Jyoti Subhash is mother of actress Amruta Subhash. They have acted together in many films (Aaji, Zoka, Gandha, Masala, Nital, Valu, Badha, Vihir) and a play (Kalokhachya Leki). She says that being together in any creative process makes the bond stronger. She played the role of Amruta's grandmother in Aaji and that of her mother in 2009 film Gandha. She also helped her while playing the role of a 60-year-old woman in her film Kavadase.^[20]